An origin for a eukaryotic lipid transfer protein fold in Asgard archaea

A phospholipid transporter in Asgard archaea sheds light on the origin of eukaryotic lipid transfer proteins | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results A phospholipid transporter in Asgard archaea sheds light on the origin of eukaryotic lipid transfer proteins View ORCID Profile Nicolas-Frédéric Lipp , View ORCID Profile Elida Kocharian , View ORCID Profile Itay Budin doi: https://doi.org/10.1101/2025.05.16.653879 Nicolas-Frédéric Lipp 1 Department of Biochemistry & Molecular Biophysics, University of California San Diego , La Jolla, CA 92093, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Nicolas-Frédéric Lipp Elida Kocharian 1 Department of Biochemistry & Molecular Biophysics, University of California San Diego , La Jolla, CA 92093, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Elida Kocharian Itay Budin 1 Department of Biochemistry & Molecular Biophysics, University of California San Diego , La Jolla, CA 92093, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Itay Budin For correspondence: ibudin{at}ucsd.edu Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract The evolution of eukaryotic cells necessitated the advent of machinery to transport phospholipids, the molecular building blocks of cell membranes, to support organelle proliferation. Eukaryotes share several classes of highly conserved lipid transfer proteins (LTP) that associate with donor membranes, bind individual phospholipids, and shuttle them to acceptor membranes. Because cells lacking organelles do not require extensive lipid transport networks, it is not known if this machinery pre-dated eukaryotic organelles or had to evolve alongside them. Here we describe a class of phospholipid transporters in the Asgard achaea that share key structural and functional similarities to eukaryotic LTPs in the START domain superfamily. Asgards contain three classes of START proteins, StarAsg1-3, which are conserved across most Asgard phyla. Of these, StarAsg1 family proteins contain the predicted structural features necessary for lipid transfer: large, hydrophobic binding pockets lined with amphipathic motifs for membrane docking. In contrast, StarAsg2 and StarAsg3 family proteins contain smaller binding cavities and minimal predicted membrane interactions. StarAsg1 from Lokiarchaeia interacts with anionic membranes both in vitro and in yeast cells, where it binds phospholipids. Lipid transfer assays show that StarAsg1 from Lokiarchaeia can exchange several classes of phospholipids between membranes, as eukaryotic START LTPs do. Structural phylogeny of START domains across the tree of life suggest that eukaryotic LTPs could share a common ancestry with StarAsg1 homologs, while StarAsg2 and StarAsg3 form a monophyletic group with a eukaryotic heat shock protein co-chaperone. We propose that the presence of inter-membrane lipid transporters in the ancestors of eukaryotic cells could have facilitated the development of complex intracellular organelles. Introduction The architecture of eukaryotic cells relies on their capacity to synthesize and transport insoluble phospholipids as the primary structural components of membranes. Organelles are bound by phospholipid membranes that are composed of distinct compositions, which tune their physicochemical properties and interactions with protein machinery. However, lipid metabolism itself is not replicated across organelles, with the endoplasmic reticulum (ER) being the primary production site for most major lipid classes. Thus, cells rely on post-metabolic transport processes to bring lipid molecules – which by their hydrophobic nature cannot diffuse through the cytosol – from the ER to all other organelles. Lipid transfer between membranes is much faster than protein trafficking 1 , 2 , and this robust transport is essential due the high lipid fluxes needed for organelles to grow and replicate. In extant eukaryotes, these processes also act to generate compositional heterogeneities between organelles that are specialized for their function 3 – 7 . Intracellular lipid transport was originally thought to occur via vesicular trafficking, but in recent years it has been discovered that many distinct lipid transfer proteins instead accomplish this task. Some LTPs are channel-shaped proteins that form bridges across contact sites, where organelles sit within tens of nanometers from each other 8 , 9 . However, most are small, soluble proteins that transiently interact with membranes and extract individual lipid molecules and transport them between membranes 10 , 11 . These shuttle-like LTPs may be tethered to membranes or contact site components, yet they still rely on soluble domains with amphipathic membrane-binding sites, regulated by dynamic bound-to-soluble switching mechanisms that support second-to-minute-scale lipid transport rates 12 , 13 . The capacity of LTPs to shuttle lipids between opposing membranes is required for the existence of organelles that define eukaryotic cells, but our understanding of the evolution of this machinery remains limited. Lipid transfer mechanisms have been identified across the periplasmic space of gram-negative bacteria involving LolA/B and MlaC homologs 14 , but no direct eukaryotic counterparts to these systems have been identified. Since all extant organelles depend on it, the acquisition of intracellular lipid transport machinery would have represented a potential bottleneck during eukaryogenesis, the evolutionary process that led to the emergence of eukaryotic cells. Eukaryogenesis occurred via endosymbiotic event(s) between 1.5 and 2.5 billion years ago 15 – 18 . It had long been accepted that eukaryotes form a sister group of archaea, with mitochondria descended from an engulfed proto-symbiont sister to alphaproteobacteria 19 – 21 . The discovery of Promethearchaeati 22 (hereafter called Asgards) as an archaeal superphylum that forms a monophyletic group with Eukarya changed our understanding of eukaryogenesis 23 and recent analyses suggest eukaryotes belong to Asgards sensu stricto 24 – 26 . Such a relationship is supported by the presence of Eukaryotic Signature Proteins (ESPs) in Asgards, including homologs of key components of eukaryotic membrane machinery: components of the Endosomal Sorting Complexes Required for Transport (ESCRT) system 27 , 28 , GTPases, Roadblock/longin families of protein, and Transport Protein Particle (TRAPP) complex 29 – 31 . Cultured Asgard, like the Lokiarchaeon Promethearchaeum syntrophicum 22 , 32 , show complex membrane topologies including tubular protrusions. Emerging molecular clock data supports a late-mitochondrial model for eukaryogenesis, in which the Asgard ancestor was already quite complex 33 . Recent imaging of exponential phase Heimdallarchaeia ( Candidatus “Yibarchaeum umbracryptum” ) also shows an abundance of large, intracellular vesicles 34 that resemble eukaryotic compartments like autophagosomes that rely on LTPs for their formation and growth 35 . These findings suggest that the complex membrane biology that characterized eukaryotic cells might have roots in Asgard archaea. The observation of internal compartments in Asgard cells suggests that they might also contain mechanisms to maintain and grow them via lipid transport, which could have been harnessed during eukaryogenesis for organelle proliferation. We thus asked if any eukaryotic LTPs classes are present in extant Asgards, which would support their presence in the ancestor of eukaryotic cells. We describe three proteins in the Steroidogenic Acute Regulatory protein-related lipid Transfer (START)-domain family in Lokiarchaeia that are widely conserved across Asgard lineages. In eukaryotes, START domains represent a widely utilized protein fold for LTPs, but also can function as regulatory proteins 36 – 38 , transcription factors 39 ,, and co-chaperones 40 . We report that one Lokiarchaeia START protein is specialized for interaction with membranes and transports phospholipids in vitro, similar to other START domain LTPs. Structural and sequence alignment methods suggest a connection between this protein family and eukaryotic START LTPs and strongly support that another Asgard START family is the origin of a conserved eukaryotic co-chaperone. Results START domain proteins in Lokiarchaeia We first examined the distribution of 14 annotated classes of shuttle-like LTPs in bacteria, archaea, viruses and eukaryotes ( Fig. 1 ). Each of these LTP classes, often defined by a family or a superfamily of proteins, contains a globular domain hosting one or two lipidic ligands in a buried binding cavity. Nine of them are exclusive to eukaryotes with very rare occurrences in other groups, suggesting that the diversity of LTPs expanded during eukaryotic evolution. In contrast, proteins containing START and Sterol Carrier Protein 2 (SCP-2) domains are distributed among eukaryotes and archaea, but also found in bacteria. From this observation, we identified potential LTPs coding sequences in the genome of the isolated Lokiarchaeia archaeon Prometheoarchaeum syntrophicum MK-D1. MK-D1 codes for one SCP-2 homolog and three START-like domains, which we named StarLok1, StarLok2 and StarLok3. We did not observe any clustering of nearby genes related to lipid metabolism or other related functions of START domain proteins. However, we focused attention on these candidates because the structural features and ability to bind hydrophobic ligands of START domains are well defined, in contrast to other families like SCP-2 41 , 42 . Download figure Open in new tab Fig. 1: Distribution and structural diversity of shuttle-like LTPs. Top panel: The proportion of total sequenced genomes that express shuttle-like LTP classes belonging to each domain of life. For each class, the InterPro accession number is given in the method section. Most known LTP classes are exclusive to eukaryotes; archaea contain examples of three (2-4). The data is split into two segments to highlight distributions under 10%. Bottom panel: Structures of example proteins from each shuttle-like LTP class show in (B) containing a bound lipid ligand (alkanes, yellow; oxygen, red; nitrogen, blue; phosphate, orange). 1. MlaC (lipid: phosphatidylethanolamine, pdb: 7VR6). 2. Lppx (α-linolenic acid and docosahexaenoic acid, 2BY0), 3. START domain of CERT (ceramide, 2E3P), 4. SCP-2 (palmitate, 4JGX), 5. RBPA (retinol, 1RBP ) , 6. Ups1 (phosphatidic acid, 4YTX), 7. BPI (phosphatidylcholine (PC), 1BP1), 8. nsLTP (palmitoleate, 1FK3), 9. Sec14 (PC, 3B7Z), 10. Osh4 (phosphatidylinositol 4-phosphate, 3SPW ) , 11. NPC2 (ergosterol, 6R4N ) , 12. GLTP domain of 4-phosphate adaptor protein 2 (FAPP2) ( N -oleoyl-galactosylceramide, 5KDI), 13. N-terminal domain of NPC1 (cholesterol, 3GKI ), 14. Lam2 (ergosterol, 6CAY). Using computational approaches, we further characterized the predicted structural features in the three Asgard START domains from the Lokiarchaeia archaeon P. syntrophicum MK-D1. We observed a charge distribution of StarLok1 that is strongly polarized, with one side featuring a large cationic patch surrounding the mouth of the protein cavity ( Fig. 2a ). This configuration suggested that StarLok1 may interact with membranes, as peripheral membrane proteins are often electrostatically polarized to interact with phospholipids 43 – 46 . A network of hydrophobic residues (F20, L26, F23, W29, F77, V90 W104, F110, F138), two opposite tyrosines (Y54, Y81), and two distant anionic residues (D21, E66), characterize the cavity of StarLok1, supporting the binding of hydrophobic ligand. Notably, the cavity lacks key polar residues generally required for enzymatic activity, such as ketone cyclization 47 . The Positioning of Protein in Membrane (PPM) algorithm suggested that StarLok1 could dock to membranes through the insertion of five hydrophobic residues, reminiscent of the binding mode of the sterol transporter STARD4 48 ( Fig. 2b ). In contrast, StarLok2 and StarLok3 have lower electrostatic polarization, residual cavities and did not show a clear mode of membrane interaction by PPM. To test the accuracy of structural predictions, we also biochemically characterized the three MK-D1 START proteins. For this, we expressed and purified recombinant StarLok1, StarLok2 and StarLok3 fused to 6xHis tag from E. coli ( Supplementary Fig. 1 ). Folding of the three proteins was confirmed by Circular Dichroism (CD) spectroscopy ( Fig. 2c ), which showed a secondary structure profile similar to those of previously-profiled START domain homologs 49 , 50 . Among the three, StarLok1 showed reduced ellipticity, StarLok2 featured an increased helical signal and reduced β-strands proportion, and StarLok3 showed a reduced number of turns. These features and the overall spectra matched those derived from the AlphaFold structures ( Supplementary Fig. 1h ), confirming the applicability of structural predictions for these proteins. Overall, structure-sequence analyses StarLok1 suggest closest homology to the plant allergen Bet v 1, while StarLok3 resembled the START domain of Aha1 ( Fig. 2d and 2e ). Download figure Open in new tab Fig. 2: START domain proteins from Promethearcheum syntrophicum have divergent structural features. a. Structural prediction of StarLok1, StarLok2 and StarLok3, represented as electrostatic surface potential ranging from −5 to +5 k B T/e and shown as a color gradient from red (negative) to white (neutral) to blue (positive). The three proteins were aligned and oriented to show the cavity entrance of StarLok1. Arrows indicate the orientation of the main α-helix of each START domain, from N- to C-terminus. b. PPM prediction of StarLok1, 2 and 3 (from left to right) docked to a model archaeal lipid membrane. The structure is colored according to the hydrophobicity of each residue from pale cyan (low hydrophobicity) to red (high hydrophobicity). Computed cavities are visualized as a 3D point cloud, color-coded by LigSite depth scores, from periwinkle blue (lower scores) to magenta (higher scores). Protein-membrane interaction energies, computed cavity sizes are provided for each protein. Key hydrophobic residues of StarLok1 embedded in the lipid monolayer are indicated with connecting lines: phenylalanine (F), leucine (L), isoleucine (I) and methionine (M). c. Circular dichroism spectra of StarLok1, 2 and 3. Experimental mean residue ellipticity (MRE in deg.cm 2 .dmol -1 ) was recorded from 190 to 240 nm (dotted lines) and secondary structures were assigned by SELCON3 decomposition (solid lines). Pie charts summarize the estimated proportion of α-helix, β-strand, turn and unordered regions for each protein. d. Left panel: Structural similarities between the AF structure of StarLok1 (yellow) and that of Bet v 1 (dark green). The crystal structure of Bet v 1 (pdb: 1BV1, light green) was also aligned. The cavity of Bet v 1 is displayed as in panel A using LigSite depth scoreq. Right panel: Structural similarities between the AF structure of StarLok3 (blue) and that of the START domain of Aha1 (Purple). The co-chaperone binding domain of Aha1 is indicated in black. RMSD of AFv4-P15494 and AFv4-O95433 aligned to StarLok1 and StarLok3, respectively, are indicated. As well, the cavity of AFv4-P15494 is reported. e. SWISS-MODEL hit template alignment of StarLok1 and Bet v 1 (yellow) and StarLok3 and Aha1 (blue) sequences. Distribution of START domains across Asgard archaea To characterize the phylogeny of START domains within Asgards, we first analyzed the relationships of 55 homologous sequences of this class across Asgard phyla. The resulting tree indicates that StarLok1 is part of a monophyletic clade comprising 21 sequences, with moderate branch support (aLRT = 0.8939) ( Fig. 3a ). Proteins of this clade (‘StarAsg1’) featured predicted binding cavities large enough (MK-D1 1595 Å 3 ; Heimdall AB_125, 1579 Å 3 ) to accommodate a wide range of lipids, including phospholipids (∼1000 Å 3 ) ( Fig. 3b ). In contrast, the clades containing StarLok2 (StarAsg2) and StarLok3 (StarAsg3) had smaller binding cavities, with StarAsg3 homologs possessing residual pockets under 300 Å 3 . StarLok2 and StarLok3 proteins, but not StarAsg1, were predicted and annotated as homologs of the activators of the heat shock protein 90 (Hsp90) ATPase (Aha1) by Protein Natural Language Model (ProtNLM/ EMBL-EBI) 51 . Thus, Asgards contain three classes of START domain proteins, represented by orthologs of StarAsg1 and StarAsg2, and StarAsg3. Download figure Open in new tab Fig. 3: Distinct clades of START domain proteins across Asgards. a. ML phylogenetic tree (PhyML Q.Pfam + R4 + F) of the START proteins identified in Asgards. Branch support values are indicated as SH-aLRT (%) and ultrafast bootstrap (%). Supports ≥ 90/90 are highlighted in green, while supports ≥ 85/60 are indicated in orange. The tree is arbitrarily rooted with a single sequence identified in the Helarchaeales (strain CR_Bin_291). Species names are color-coded according to their taxonomic groups: Helarchaeales (magenta), Heimdallarchaeia (wine), Thorarchaeia (orange), Lokiarchaeia (blue), except for MK-D1 (green). Sequence accessions and lengths corresponding to the monophyletic clade of StarAsg1 are highlighted in yellow, while the monophyletic clade of StarAsg2 and StarAsg3 is highlighted in purple. b. Comparative analysis of cavity sizes in StarAsg1 and its homologs in Asgards. The Uniprot accession numbers are listed along the y-axis, with the corresponding cavity sizes represented by horizontal bars. Yellow bars indicate proteins belonging to the StarAsg1 clade, and purple bars indicate proteins from the StarAsg2 and StarAsg3 clades. ND indicates the absence of a detectable cavity. c. Abundance of the START domain classes in different Asgard phyla. The dot size is proportional to the number of identified START domains per species; an empty circle indicates the absence of START domains in that phylum. The average number of determined species in the dataset is given next to each phyla’s name in parenthesis. d. Abundance of the START domains identified in a representative set of archaeal, bacterial and eukaryotic species referenced in the UniProt database. The number of species in each clade is indicated in parentheses. The set corresponds to that used for the analyses in Fig. 6 . In a holistic analysis of Asgard proteomes, we identified 411 START domain sequences distributed across 203 current species. We did not observe any START homolog in several Asgard phyla, including Odin and Wukong. However, the three START domains clades (StarAsg1-3) are well represented in most phyla: Kari, Hod and Heimdall phyla all display the four classes of START domain, including StarAsg1 ( Fig. 3c ). We also identified three occurrences of DAPG hydrolase (PFAM 18089) as well as twenty-nine Rieske [2Fe-2S] type-protein diverging from a group of START domains, which we named StarAsg4 ( Fig. 3c and Supplementary Fig. 2). Overall, this analysis suggests a distinction between StarAsg1 (SH-aLRT; UFBoot = 83.3; 83), StarAsg3 (73; 81) and the phylum including StarAsg4 and StarAsg2 (86.1; 71) ( Supplementary Fig. 2). StarLok1 binds anionic membranes Our computational analyses suggested that among the three Lokiarchaeia START domain proteins, StarLok1 is uniquely suited for membrane binding. Consistent with this model, StarLok2 and StarLok3 showed high solubility, but StarLok1 aggregated when concentrated in the absence of detergents ( Supplementary Fig. 3 ). Detergent-free StarLok1 could be stabilized at acidic pH, which promotes increased cationic electrostatic repulsion along the amphipathic protein face, and was additionally stabilized by acetate ( Supplementary Fig. 3c-e ). StarHod1, a close ortholog of StarLok1 present in Hodarchaeales ( Fig. 2a ), showed similar predicted structural features ( Supplementary Fig. 4a ) and stabilization by acetate (Supplementary Fig. 4b) . Asgards, including Lokiarchaeia, features an acetogenic metabolism, in which carbon dioxide is fixed into acetyl-CoA via the Wood-Ljungdahl carbon fixation pathway 52 . Such organisms produce acetate as a byproduct of ATP from acetyl-CoA 53 , which might thus influence oligomerization or other interactions of StarAsg1 proteins. We next assayed the interaction of each Lokiarchaeia START protein with lipid membranes. Using a dilute solution of each protein, we measured changes in light scattering upon incubation with liposomes containing the neutral lipid di-oleoyl-phosphatidylcholine (DOPC) and the anionic lipid phosphatidylserine (PS). Addition of StarLok1 triggered clustering of liposomes containing 5% or higher PS concentrations, which were minimized in the presence of neutral DOPC liposomes ( Fig. 4a ). In contrast, StarLok2 and StarLok3 showed minimal interactions with PS-containing liposomes ( Fig. 4b ). We could also detect gravity separation of StarLok1 in solutions with PS-containing liposomes, but not DOPC liposomes (Supplementary Fig. 5g ). StarLok1 also showed features consistent with its hypothesized hydrophobic nature, including aggregation in the absence of liposomes ( Supplementary Fig. 3 ), high levels of staining with hydrophobic dyes on SDS-PAGE ( Supplementary Fig. 5c ), and accumulation at air-water interfaces in density-based separation experiments ( Supplementary Fig. 5g ). Download figure Open in new tab Fig. 4: StarAsg1 proteins are specialized for interaction with anionic membranes. a. DLS of StarAsg1 with liposomes of different lipid compositions. Light scattering was recorded for 2 min after the addition of 50 µM liposomes composed of pure DOPC, or DOPC doped with 5 mol% POPS, 10 mol% POPS, or 30 mol% POPS. At t = 2 min, 1 µM StarAsg1 was added to the mixture, and measurements were acquired for an additional 20 min. Increasing POPS causes protein-mediated aggregation of the liposomes. Addition of StarLok2 and StarLok3 addition to DOPC liposomes with 30 mol% POPC does not induce aggregation. b. StarLok1-GFP and StarHod1-GFP expressed in budding yeast binds the PM. The lower panels are intensity heat maps of the corresponding confocal image to highlight the polarized distribution of StarAsg1 in membranes surrounding the yeast bud neck and budding daughter cells. c. StarLok1 localization and polarization correlates with PS abundance in the OM. PS is detected with the protein sensor mRFP-C2 LACT as previously descibed 47 . Corresponding intensity heatmaps are reported as in b. The lower left composite image of StarLok1-GFP (cyan) and mRFP-C2 LACT (magenta) was used to determine colocalization shown on the left curve. An increase of signal from “i” to “ii” across the PM is indicated by an arrow. Scale bar, 2 µm. We also asked if StarAsg1 proteins can interact with cell membranes. For this, we expressed StarLok1, Lok2, Lok3 and Hod1 fused to a C-terminal GFP in Saccharomyces cerevisiae . Visualization of StarLok1-GFP in yeast cells revealed a strong plasma membrane (PM) association with additional enrichment at regions surrounding the bud neck of cells. More specifically, StarHod1 localized exclusively in the PM in a polarized manner ( Fig. 4b , Supplementary Fig. 6 ). This distribution echoed previous observation for a PS-binding sensor, the C2 domain of lactahedrin (C2 LACT ) in yeast cells 54 . Expression of the mRFP-C2 LACT sensor in StarLok1-GFP expressing cells confirmed colocalization of StarLok1 with membranes enriched in PS. These experiments suggest that StarAsg1 proteins have specialized for binding to anionic membranes in cells. In contrast, StarLok2 and StarLok3 remained soluble in the yeast cytosol ( Fig. 4c ). StarLok1 binds phospholipids and transfers them between membranes Given that StarLok1 interacts with membranes in vivo, we asked if it also extracts lipid ligands when expressed in cells, an established means to identify ligands of lipid transfer proteins 55 . We isolated GFP-tagged StarLok1, 2, and 3 and StarHod1 from yeast cell pellets using affinity purification with beads displaying anti-GFP antibodies ( Fig. 5a ). We then extracted any bound lipids from protein displaying beads and analyzed their composition and abundance by LC/MS 2 (lipidomics). While StarLok2 and StarLok3 showed low lipid binding, StarLok1 and StarHod1 extracts contained nearly equimolar bound phospholipid (110 pmol) to the capacity of bound proteins on the beads (150 pmol). Lipidomics showed that StarLok1-bound ligands were enriched in PS, while StarHod1 ligands were enriched in phosphatidylcholine (PC) ( Fig. 5b ). Both also bound phosphatidylethanolamine (PE). In contrast, lipids extracted from StarLok2 and StarLok3 samples were of lower abundance, and contained predominantly phosphatidylinositol (PI) and to a lesser extent PC, reflecting background whole cell phospholipids 56 . These results indicated that StarAsg1 proteins extract specifically phospholipids from cell membranes. Download figure Open in new tab Fig. 5: Characterization of lipids bound to StarAsg proteins and lipid transfer activity. a. Schematic representation of the experimental workflow used to identify lipids bound to GFP-tagged StarAsg proteins expressed in yeast and plot showing total lipid abundance quantified by LC/MS 2 . b. Abundance of phospholipid classes associated with StarLok1, StarHod1, and StarLok2/3. StarAsgard1 proteins show increased levels of PC, PE, and PS binding. c . Schematic of lipid transfer assay. donor liposomes (L D , 200 µM total lipid) And acceptor liposomes (L A , 200 µM total lipid) were incubated with 5µM of StarLok1 in PBS buffer (pH 7.4). d. NBD-lipid transfer kinetics of StarLok1 (yellow) incubated with L D and L A liposomes compared to a blank control containing an equivalent amount of N-lauroylsarcosine (gray), or an equivalent molar amount of proteinase K–inactivated StarLok1 with (purple). The fluorescence signal was normalized using calibration control to yield the absolute amount of NBD-lipid transferred (µM) and is represented as the mean of n independent replicates ± SD. Each plot represents a different NBD-tagged ligand, whose structure is shown to the right. e. Calculated lipid transfer rate derived from the initial slopes of the transfer curves (mean ± SEM). Statistical significance was determined using two-way ANOVA followed by Tukey’s multiple comparisons test. ** P < 0.01; **** P < 0.0001. Based on its capacity to bind phospholipids in vivo, we asked if StarLok1 could act as a bona fide phospholipid transporter. The movement of NBD-labeled phospholipid analogues from donor liposomes can be quantified using Förster resonance energy transfer (FRET) to Rhodamine-PE that is bound to acceptor liposomes ( Fig. 5c ), as previously described for START LTPs such as STARD2, STARD10, PITPα/β and CERT 57 – 59 . Upon addition of detergent-solubilized StarLok1 to donor liposomes containing NBD-PS, NBD-PC, or NBD-PE, a robust increase in FRET was observed, indicating lipid transfer ( Fig. 5d ). These three substrates correspond to the phospholipids enriched in StarLok1-GFP extracts compared to background binding in StarLok2/3-GFP samples ( Fig. 5b ). Phospholipid transport was not observed beyond baseline when equivalent detergent was added, and when the protein was digested with Proteinase K ( Supplementary Fig. S7a ), the specific lipid transfer was drastically inhibited. Using equilibrated liposomes, we quantified initial transport rates ranging between 1-2 lipids·min -1 per StarLok1 for these phospholipids ( Fig. 5e ). We also observed that transport NBD-labeled ceramide, a lipid that lacks a phosphate headgroup, was slower than that of the phospholipids, while transport of the FRET acceptor Rhodamine-PE, which contains a very large headgroup, was not detected in control liposomes ( Supplementary Fig. S7b-c ). Sequence and structural phylogeny of START domains across the tree of life START domains are well represented across the tree of life, including in non-Asgard archaeal lineages (Euarchaeota, TACK group, DPANN group, Thermoplastada) ( Fig. 3c ). Given their universal nature, we sought to understand the phylogenetic relationships across START domains and test the likelihood that eukaryotic LTPs and Hsp90 co-chaperones originated from Asgards. For this, we manually curated a set of 161 representative taxa containing eukaryotes, archaea, and bacteria across the tree of life ( Fig. 6b ). This yielded a dataset containing 748 START domains containing sequences We applied both sequence-based Maximum Likelihood (ML) approaches and structural homology analysis to generate trees. For the former, we screened several sequence alignment algorithms to optimize the overall conservation score of different sequence subsets and generated several ML trees ( Supplementary Fig. 8 to Fig. 14 ) based on subsets of the dataset to address specific questions. For the latter, we employed the FoldTree 60 algorithm, which generates trees based on structural divergences measured by Foldseek 61 , on the whole dataset. Because START domains often exist in tandem repeats in eukaryotic genomes or as parts of multi-domain proteins, we developed a tool (ProtDomRetriever) to isolate and align individual domains ( Fig. 6b ). While the conclusions were consistent across both approaches, FoldTree analysis revealed the evolutionary history of the START domain with deeper resolution and accurately identified the evolutionary history of known taxa ( Supplementary Fig. 15 and 16 ). This is likely due to the high sequence plasticity in START domains – less than 25% of proteins passed the Chi2 composition test – that resulted in high entropy and gap rich sequence alignments that are challenging for ML analysis. However, the overall structural fold remains constant in this superfamily, making it well-suited for structural homology. Download figure Open in new tab Fig. 6: Sequence and structural domain phylogeny supports orthology between StarAsg proteins and eukaryotic START domains. a. ML phylogenetic tree inferred from 227 START domain sequences, including StarAsg proteins (see Fig. 3a ), representative eukaryotes ( Homo sapiens, Nematostella vectensis , Capsaspora owczarzaki , Dictyostelium discoideum , Thecamonas trahens , Guillardia theta ), 11 archaeal species, and 25 bacterial species. Sequences were aligned using MAFFT (L-INS-i; --localpair --maxiterate 1000) with BLOSUM80 and modified gap parameters (--op 1.53 --ep 0.5 --bl 80 --lop −1.0 --lexp −0.5). The best-fit substitution model was selected using ModelFinder according to BIC (Q.PFAM+FO+R5), and ML inference was performed with IQ-TREE. Branch support values correspond to SH-aLRT (%) and ultrafast bootstrap (%) from 1,000 replicates each and are shown in magenta for major clades only. Branch lengths represent substitutions per site. The tree is rooted using Shigella flexneri and Escherichia coli . Branch-tip labels are colored according to taxonomic origin (brown, eukaryotes; blue, archaea; red, bacteria). Yellow, purple, and gray stripes adjacent to branch-tip labels indicate Asgard sequences belonging to the StarAsg1, StarAsg2/3, or undefined clades, respectively (see Fig. 3a ). Red branches highlight the monophyly of mitochondrial CoQ-binding protein 10 homologs with bacterial RatA. Purple, yellow, and brown branches denote StarAsg2/3 and AHA1, StarAsg1, and eukaryotic START LTP clades, respectively. b. Workflow for large-scale structural domain phylogeny. ProtDomRetriever was used to identify START domain boundaries using InterPro superfamily accessions, CATH/gene3D annotations, and SUPERFAMILY HMM library accessions. Corresponding structural domains were extracted from AlphaFold models and phylogenetic inference was performed using the FoldTree pipeline. c. Rooted structural phylogenetic tree of 805 START domains from 161 species. Visual annotations (branch colors, label colors, and stripe coding) follow the scheme described in panel a . Each monophyletic START domain class is numbered. Nodes are represented as black dots. The scale bar indicates substitutions per site. The bottom right graph indicates abundance of the START domains identified in representative archaeal, bacterial and eukaryotic species referenced in the dataset database. The number of species in each clade is indicated in parentheses. d. Simplified cladogram derived from panel. LTPs previously characterized in vitro are highlighted in yellow, signifying the major eukaryotic START domain protein families. Monophyletic classes are labeled using the same numbering as in panel c. All our trees estimated an ancient origin to the START-domain that preceded eukaryogenesis. Sequence alignment-based ML phylogeny ( Fig. 6a ) and structure-based trees ( Fig. 6c ) summarized by a pruned cladogram ( Fig. 6d ) showed a distinct branching group consisting of mitochondrial Coenzyme Q-binding proteins (COQ10-like class) and Ribosome association toxin A (RatA) in bacteria, consistent with previous groupings 62 . The divergence of COQ10 from alphaproteobacterial RatA homologs was also supported by strong branch support (aLRT 0.947; UFBoot 0.94) in ML trees ( Fig. 6a ). These analyses reflect a likely bacterial origin of the nucleus-encoded but mitochondrial-localized COQ10 binding protein. Our analyses reveal an archaeal origins of co-chaperones of Hsp90 (Aha1, ASHA1, ASHA2). Phylogenetic analyses indicate that Aha1-like proteins originate within Asgards and form a monophyletic group including nine StarLok3 asgard homologs: StarAsg3, which diverged from StarAsg2 clade in Asgards. The grouping of AHA1 and StarHod3 is strongly supported (SH-aLRT; UFBoot = 0.948; 0.9) and this clade is within the broader StarAsg3 lineage (0.938; 0.79) ( Fig. 6a ). In the full dataset, StarAsg3 and AHA1 consistently form a monophyletic group with StarAsg2-like proteins, albeit with low support ( Fig. 6a , Supplementary Fig. 8 ). In contrast, when restricting the analysis to prokaryotic START domain only, StarAsg2 and StarAsg3 form a strongly supported monophyletic clade (0.975; 0.92; Supplementary Fig. S13 ), consistent with earlier analyses ( Fig. 1a ; Supplementary Fig. 2) . We identified several bacterial START domains with structural similarity to StarAsg2 (but not StarAsg3) ( Fig 6c ), which also cluster weakly with StarAsg2 in ML trees ( Supplementary Fig. 14 ). However, to our knowledge, no experimental evidence supports interaction between bacterial START proteins and Hsp90. Thus, AHA1-like proteins are likely to have been inherited from an ancestral StarAsg3 lineage in Asgards prior to the emergence of the first eukaryotic common ancestor. In eukaryotes, START domains are the basis for multiple classes of LTPs that shuttle lipids intracellularly. All the LTP START domains in our dataset find their roots with protein sequences in Heimdallarchaeia (A0A1Q9NB21, A0A1Q9P3Z5, A0A1Q9NZ53 and A0A1Q9NLH4), which belong to the StarAsg1 clade. Although the available phylogenetic signal does not allow a definitive assignment of START domain LTPs to an Asgard origin, several ML reconstructions and the FoldTree analysis consistently position Heimdallarchaeia StarAsg1-like sequences near the base of eukaryotic LTP diversity. In the global dataset ( Fig. 6a ), StarHod1 (A0A1Q9NB21), together with A0A1Q9P3Z5, A0A1Q9NZ53 and A0A1Q9NLH4, forms a monophyletic group associated with eukaryotic START LTPs. In alternative reconstructions, individual StarAsg1 sequences cluster within the eukaryotic LTP assemblages ( Supplementary Fig. S10 ). When restricting the analysis to eukaryotic START LTPs and StarAsg1 homologs (Supplementary Fig. 9) , StarAsg1 sequences form a well-supported clade (97.9; 89) together with Bet v 1-like proteins, including plant PYL1; this combined assemblage connects to the broader STARD-like LTP and PITP radiation, albeit with weaker support (58.5; 53). All StarAsg1-like sequences belong to a group characterized by an enlarged predicted binding cavity, a structural feature that is enhanced in eukaryotic START LTPs (Supplementary Fig. 17a) . Structurally related START proteins were identified in other archaeal lineages ( Fig. 3d ), several of which grouped with StarAsg proteins in Foldseek-based structural analyses (Supplementary Fig. 17b) ; however, these non-Asgard proteins display smaller or poorly defined predicted binding cavities (Supplementary Fig. 17c-d) , suggesting that they may not function as LTPs. Based on these analyses and the established relationship of eukaryotes within Asgard archaea, a parsimonious grouping is that eukaryotic START domain LTPs diversified from an inherited StarAsg1 protein. Two alternative hypotheses – that START domain LTPs diversified from StarAsg2/3 ( Supplementary Fig. 11 ) or from bacterial-derived START domains ( Supplementary Fig. 12 ) – are not supported by ML trees. Horizontal gene transfer of an ancestral START domain LTP to eukaryotes is also unlikely unless missing prokaryotic phyla contributed to eukaryogenesis, as we did not detect taxonomic clades branching in eukaryotic LTPs. Discussion Trafficking of phospholipids, given their negligible solubility, is a hallmark of eukaryotic cells and is a prerequisite for the stable coexistence of multiple membrane organelles. Identification of LTPs within Asgard would support a model in which eukaryotic ancestors already contained proteins supporting this key eukaryotic function. To this end, we described three conserved START domain proteins in Lokiarchaeia, the same fold that forms the basis for a large and diversified group of eukaryotic LTPs. One, StarLok1, binds phospholipids and transports them between membranes. It is a member of a large clade of Asgard START domains (StarAsg1) that contain predicted structural features found in LTPs: a large, hydrophobic binding pocket lined with an amphipathic and basic region for interaction with anionic lipid bilayers. Phylogenetic and structural homology analysis suggests a broader relationship between Asgard START proteins and those in eukaryotes. Structural and sequence alignments were able to generate a putative tree of START domains across kingdoms suggested that STARD-like proteins and similar eukaryotic LTPs emerged from an ancient START domain fold, which could have been present in Asgards as StarAsg1 homologs. A second class of Asgard START domains, StarAsg3, includes ancestral proteins for Activator of Heat Shock Protein 90 (Hsp90) ATPases (AHAs), an essential co-chaperone for protein stability and maturation in eukaryotes. Another class of START domain proteins conserved in eukaryotes, the Coenzyme-Q binding protein 10, shares a common ancestry with bacterial Ribosome association toxin A proteins and was thus likely inherited from the alphaproteobacterial ancestor of mitochondria, consistent with previous reports 62 , 63 . To specialize for function as an LTP, protein families like START domains would need to adopt enlarged hydrophobic binding cavities lined by a membrane-interacting motif, and that is the feature we observe in StarAsg1 proteins compared to their sister groups StarAsg2/3. More broadly, we observe that an increase in binding cavity size is a general feature for the specialization of START domains for lipid binding across both Asgards and eukaryotes ( Supplementary Fig. 17a ). Our phylogenetic analysis also supplements previous classifications of START domain LTPs within eukaryotes 42 , 64 , 65 with several additional details ( Fig. 6d ). These include orthology between STARD4/5/6 and STARD1/3; both of which radiate independently in animals and their closest unicellular relatives, like Capsaspora owczarzaki . Similarly, the two START domains containing acyl-CoA thioesterases (STARD14 and STARD15) also show orthology and arose prior to the divergence of animals and C. owczarzaki . More distantly, CERT (STARD11) is grouped as an independent class. We also observed orthology between STARD8, STARD12, and STARD13, all of which contain a START domain fused to a small GTPase activating protein Rho domain, which are also common to choanoflagellates. We observed an earlier separation of STARD10 orthologs from an ancestral EDR2-like START domain in plants, with occurrence in a broader group of unicellular eukaryotes, however the position of the STARD10 group remained ambiguous. Finally, our tree suggests that STARD2 (PCTP) and STARD7 form a paralogous sister group from other members of the STARD group. Finally, the PITPs group, which are phosphatidylinositol and phosphatidic acid transporters 66 , consistently diverged from STARD proteins at the earliest stage of START LTPs radiation in eukaryotes, supporting the acquisition of phosphatidylinositol as key lipid in eukaryotes 67 . Recent advances suggest that lipid transport could be relevant in Asgard cells, even if they lack complex organelles that evolved during eukaryogenesis. Heimdallarchaeia have been recently observed to contain large intracytoplasmic vesicles that could require lipid transport activity to support 34 . Importantly, bound ligands and transported substrates of StarLok1 include PS, a phospholipid with an equivalent in archaea archaeatidylserine synthase (AS) 68 . Lokiarchaea MK-D1 contains a phosphatidylserine synthases (PSS) homolog (UniProt: A0A5B9DD23) containing key residues for serine binding ( Supplementary Fig. S18) and thus is likely to natively contain phospholipids with an identical polar group as PS. Although archaeal phospholipids most commonly feature an opposite chirality to that of those found in eukaryotes, this aspect is unlikely to be a hindrance for LTPs that bind ligands based on cavity size and hydrophobicity, and ionic interactions with headgroups features. Several aspects of Asgard lipid biology are still being investigated, but AS and other phospholipids represent a likely in vivo substrate for StarLok1. Both StarLok1 and StarHod1, another StarAsg1 protein in Hodarchaeala, also bind the plasma membranes in yeast cells, which are enriched in PS, as well as other anionic phospholipids. The ubiquitous polarity of StarAsg1 proteins, containing basic patches near their ligand binding pocket, suggests that an anionic inner membrane is widespread in Asgards, where it could be supported by the activity of AS synthase and putative archaeatidylinositolphosphate synthases (A0A5B9DAH1) 32 . A highly anionic cytosolic leaflet and more neutral outer leaflet is a hallmark of eukaryotic cells that dictates the properties of membrane-associating proteins, like LTPs. It is possible that this membrane feature also has its origins in archaeal ancestors, given the noted asymmetric nature of archaeal bolalipids that contain anionic phosphoheadgroups on the cytosolic leaflet and neutral glycosylations on exoplasmic leaflets 69 . In this model, eukaryotic cytosolic proteins that interact with membranes, like LTPs, might have originated from the archaeal ancestor, even if their backbone and phospholipid tails differ. Additionally, the asymmetric distribution of anionic lipids is essential for phagocytosis and vesiculation in eukaryotes 70 – 73 , thus the presence of anionic lipid in the cytosolic leaflet of Asgard membranes could be relevant for the intracellular vesicles observed in Asgards 34 . The first eukaryotic LTPs could have acted as metabolic facilitators by fortuitously transferring newly synthesized lipids between membranes, enabling the proliferation of intracytoplasmic compartments. Ancestral START LTPs could have fulfilled this function with their large, hydrophobic binding cavity and membrane-binding capacity. Later, the emergence of more sophisticated LTPs capable of coupling the transfer of multiple lipids, such as the Osh/ORP proteins that counter-exchange lipid ligands for PI(4)P 74 would have enabled the generation of heterotopic organelle membranes with specialized lipid compositions. Bridge-like LTPs, such as those in the VPS13 class, could have emerged due to the need for rapid membrane proliferation in emerging aspects of eukaryotic cell biology, including mitochondrial network extension 75 , the generation of autophagosomes 76 or spores 77 , and were later adapted to support routine lipid transfer in larger, increasingly compartmentalized cells, in which contact sites emerged as central lipid transfer hubs 78 , 79 . In this way, the increasing complexity of eukaryotic cell biology could have been enabled by biochemical advances in the ability of proteins to transport lipids. Further integration of evolutionary and biochemical approaches could help resolve how adaptations in cellular architecture and lipidomes co-arose during eukaryogenesis. Data Availability Alignments and trees generated are available at iTol ( https://itol.embl.de/shared/BudinLab ), scripts used at GitHub ( https://github.com/NicoFrL/ProtDomRetriever and https://github.com/NicoFrL/MAFFT_ScoreNGo ). Data files used to generate figures will be made available on FigShare. Competing interests The authors have no competing interests to declare. Materials and methods Nomenclature START: StAR-related transfer, it refers to START-like domain superfamily stricto sensu (IPR: IPR023393 ). In opposition to other “Starkin” domains such as members of VAD1 Analog of StAR-related lipid transfer family and PRELI/MSF1, both display a helix-grip fold analog to START domains. StAR: Steroidogenic acute regulatory protein. SRPBCC: NCBI Conserved Protein Domain superfamily associated to the START domain (NCBI: cl14643), START/RHO_alpha_C/PITP/Bet_v1/CoxG/CalC ligand-binding domain superfamily. STARD: (StAR)-related lipid transfer domain. Lipid transfer protein classification analysis The 14 classes of LTP we identified were previously described 80 – 84 . The Interpro accession number of each of this class was used to determine the number of taxonomic species expressing at least one of these proteins: 1) Tgt2/MlaC (Superfamily IPR042245), 2) LolA/LolB/Lppx (Superfamily IPR029046), 3) START-like domain (Superfamily IPR023393), 4) SCP2 sterol-binding domain (superfamily IPR036527), 5) Calycin-Lipocalin (superfamily IPR012674), 6) PRELI/MSF1 (Domain IPR006797), 7) LBP/BPI/CEPT (i.e. Bactericidal permeability-increasing protein, alpha/beta domain; Superfamily IPR017943), 8) nsLTP (i.e. Bifunctional inhibitor/plant lipid transfer protein/seed storage helical domain; Superfamily IPR036312); 9) CRAL/Trio (Superfamily IPR036865), 10) ORP/Osh (Superfamily IPR037239), 11) GM2-AP (Superfamily IPR036846), 12) GLTP (Superfamily IPR036497), 13) NPC1 N-ter (i.e. Niemann-Pick C1, N-terminal; Domain IPR032190), 14) VASt (i.e. VAD1 Analog of StAR-related lipid transfer; Domain IPR031968). Expression and purification of StarLok1 A codon optimized 444 bp cDNA coding for StarLok1 (UniprotKB: A0A5B9D5D6) C-terminally fused to a thrombin cleavage site (LVPRGS) followed by a 6xHis tag was synthesized and cloned into a pET-21a(+) vector (GeneScript) and expressed in BL21-Gold(DE3) (Agilent). StarAsg1 was purified either from native soluble fraction or refolded from an insoluble fraction. For native purification, a 1 L culture LB Lennox with 50 µ g/mL ampicillin was grown to an OD 600 of 0.5 at 37°C then induced (0.2 mM IPTG) before overnight growth at 16°C. Cell pellets were resuspended in cold TN buffer (50 mM Tris, 500 mM NaCl, pH 8.0, at 4°C) supplemented with protease inhibitor cocktail (Roche cOmplete EDTA-free, 10 µM bestatin, 1 µg/mL pepstatin A, 10 µM phosphoramidon), 10 mM imidazole, 1 mM DTT, 20 µg / mL of DNase I (Roche), 5 mM MgCl 2 and 1 mg/mL of lysozyme. Cells were then disrupted by sonication and the lysate was clarified by ultracentrifugation (186,000 x g, 1h, 4°C). Fifty volumes of supernatant were applied to one volume of Ni-NTA resin slurry (cOmplete His-Tag Purification Resin) and incubated overnight at 4°C. The resin suspension was applied to an Econo-Pac chromatography column (Bio-Rad) successively washed with TN containing 10 mM and 20 mM imidazole. The protein was eluted with TN containing 250 mM imidazole and the eluate concentrated at 2500 x g using Amicon Ultra centrifugal unit (MWCO 3000). The protein was further purified by SEC on an Akta pure 25 FPLC with a Sephacryl S200 HR column equilibrated with TN buffer at a flow rate of 1mL/min and fractionation volume of 2.5 mL. Glycerol was added to the buffer (10% v/v) and protein-containing fractions were pooled and concentrated up to 20 µ M. Concentration was determined by both A 280 (DeNovix DS-11 spectrophotometer) and Sypro Orange (ThermoFisher) staining of SDS-PAGE using BSA standards and samples were frozen in liquid N 2 and stored at −80°C. For refolding of insoluble protein, StarLok1 expression was performed as described in the previous section with minor modification. When OD 600 reached 0.6, 1 mM IPTG was added into the culture that was then incubated three hrs at 37°C before collecting the cells and preparing the pellet for freezing. Pellets were resuspended in 50 mM HEPES-KOH pH 7.00 with a protease inhibitor cocktail (Roche cOmplete EDTA-free, 10 µM bestatin, 1 µg/mL pepstatin A, 10 µM phosphoramidon) and passed 4 times through a high-pressure homogenizer at 1000 bar (EmusiFlex-c3, Avestin). 20 µg/mL of DNase I (Roche), 5 mM MgCl 2 were added, and the inclusion bodies were then pulled down by ultracentrifugation (186,000 x g , 1 hr, 4°C). The supernatant was discarded, and the white pellet was solubilized in HU buffer (50 mM HEPES-KOH pH 7.0 with 6M urea) by mild vortexing and pipetting and one passage through high-pressure homogenizer (1000 bar). After centrifugation (186,000 x g, 1 hr at 4°C), 10 mM imidazole was added to the clear supernatant, which was applied to 50:1 to Ni-NTA resin slurry (cOmplete His-Tag Purification Resin) and incubated 3 hr at 21°C. The suspension was applied to an Econo-Pac chromatography column (Biorad), washed twice with 10 bed volumes of HU with 20 mM imidazole, and eluted with eight bed volumes of HU with 250 mM imidazole. Refolding was performed by dialysis (3.5K MWCO, Slide-A-Lyzer™ MINI Dialysis Devices) twice against 150 volumes of acetate buffer (164 mM acetic acid (OHAc), 36 mM sodium acetate (NaOAc), pH of 4.04) over 14 hr. Screening for refolding conditions was initially done by light microscopy (ThermoFisher EVOS) after low-volume dialysis. Refolded samples were further purified by FPLC on a HiPrep 16/60 Sephacryl S200 HR column (Cytiva) equilibrated with acetate buffer at a flow rate of 1 mL/min and fractionation volume of 2.5 mL. Purification and refolding were monitored by SDS-PAGE. This protocol yielded 5 to 7 mg of protein per 1 L of bacterial culture, as determined by A 280 . For detergent solubilized protein used in lipid transfer assays, expression in E.coli BL21 was induced with 1 mM IPTG once the OD 600 reached 0.95 and incubated for 3 hr. Bacterial pellets were collected as previously described. Bacterial pellets were resuspended in PBS with 1% N-lauroyl-sarcosine (NLS) and 10 mM imidazole, 5 mM MgCl2 and 20 µg / mL of DNAse I. Bacteria were lysed by sonication and the lysate was clarified by ultracentrifugation (186,000 x g , 1 hr, 4°C). The supernatant was then applied to a Ni-NTA resin slurry and incubated for 3 hr. After two successive washes (PBS, 1% NLS, 10 mM imidazole), StarLok1 was eluted with 250 mM imidazole. The eluate (12 mL) was adjusted with PBS buffer and no detergent, and concentrated to 2 mL using an Amicon Ultra centrifugal unit (MWCO 3000), diluted again to 13 mL with PBS buffer and concentrated once more until the solution started to oil-out around 500 µL. The protein concentration (1.53 mM) was determined by absorbance spectrometry at 280 nm. The sample was stored at 4°C, and purity and stability was determined by SDS-PAGE prior NBD-lipid transfer assay. Expression and purification of StarLok2 and StarLok3 A codon optimization 489 bp cDNA coding for StarLok2 (UniprotKB: A0A5B9DET0) and 432 bp coding for StarLok3 (UniprotKB: A0A5B9DAG8), each N-terminally fused to a 6xHis tag followed by a thrombin cleavage site (LVPRGS), were synthesized and cloned into a pET-28a(+) vector (GeneScript) and expressed in BL21-Gold(DE3) (Agilent). Each protein was purified from 1 L culture in its native form as described for StarLok1 in TN buffer supplemented with 1 mM DTT. In this standard condition, the protocol yielded more than 10 mg of protein per 1 L of bacterial culture. Protein concentration and purity were controlled by SDS-PAGE stained with InstantBlue (Abcam). Circular Dichroïsm CD was performed on a Jasco J-1500 spectrometer using a 350 µL synthetic quartz glass cell with a 1 mm path length (CV1Q035AE2, ThorLabs). Measurements were taken of StarAsg1 in either 20 mM Tris, 120 mM NaF, pH 7.4 (native), or in a solution prepared by diluting the stock refolded protein (HOAc/NaOAc, pH 4.0) in milli-Q water. Each spectrum represents the average of five continuous scans recorded from 185 to 260 nm with the following parameters: bandwidth of 1 nm, step size of 0.1 nm, scan speed of 50 nm·min−1, CD scale of 200 mdeg/0.1 dOD, and digital integration time (D.I.T) of 4 s. A control spectrum of buffer samples without protein was subtracted from each protein spectrum. For temperature interval scans, an equilibration time of 20 s was set before each scan at 10°C intervals. The measured ellipticity was converted to molar ellipticity, considering the cuvette path length and the mean residue concentration of proteins. For secondary structure determination, the spectra were analyzed in the 185–240 nm range using the SELCON3 algorithm and the SP175t dataset on the DICHROWEB server 85 and compared with the algorithm available on the BeStSel web server 86 . Thermal denaturation results were displayed using matplotlib. Protein structure analysis Structures were rendered using Pymol (v2.5.8, Schrödinger, LLC). Structure alignment comparisons were conducted using the cealign algorithm and the RMSD values were reported. Model-template alignments were obtained using SwissModel 87 . Cavities point clouds were calculated using the CavitOmiX plugin (v. 1.0, 2022, Innophore GmbH) 88 and cavities were calculated using a modified LIGSITE algorithm 89 . To compute the cavity size, a script calling CavFind and LigSite modules from CavitOmiX was used to extract the number of cavities, their respective volumes and ligsite score. Protein electrostatic surface potential was determined using APBS (pdb2pqr) 90 with a grid spacing of 0.26. Hydrophobicity was determined using the Eisenberg scale 91 . The Position of Protein on Membrane (PPM) was determined using PPM 3.0 web server 92 , using a flat theoretical archaeal plasma membrane composed of Phosphatidylglycerol, Cardiolipin, Archaeol and Menaquinone 8 at a ratio 64:7:8:21. Changing the membrane composition did not significantly change the obtained results. The AlphaFold2 structure of StarLok1 with a thrombin cleavage linked to 6x(His)-tag was computed using ColabFold v1.5.5 notebook. The secondary structure composition was determined using either STRIDE 93 and DSSP algorithm 94 . The theoretical CD spectra of StarLok1 was determined using either SESCA 95 and PDB2CD 96 for comparison with experimental measurement. Protein mass spectrometry StarLok1 sample purified in native condition was dialyzed three times (1 hr, overnight, and 1 hr) against 1 L of TN buffer (10 mM Tris-HCl, 50 mM NaCl, pH 7.4) using a 0.5 mL Slide-A-Lyzer cassette, to ensure complete glycerol removal. For StarLok1 purified in acetate buffer, no dialysis was performed.. Protein samples were analyzed for purity and mass identification by liquid chromatography with an Agilent 6230 time-of-flight mass spectrometer (TOFMS) with JetStream electrospray ionization source ESI (LC-ESI-TOFMS) Liposome preparation Stock solutions of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1-palmitoyl-2-{12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl}-sn-glycero-3-phosphoserine (16:0-12:0 NBD-PS), 1-palmitoyl-2-{12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl}-sn-glycero-3-phosphoethanolamine (16:0-12:0 NBD-PE), 1-palmitoyl-2-{12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl}-sn-glycero-3-phosphocholine (16:0-12:0 NBD-PC), N-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-D-erythro-sphingosine (C12-NBD-Ceramide) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (Rhodamine-PE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS) in chloroform (Avanti Polar Lipids) were mixed at the desired molar ratio. The solvent was evaporated under vacuum (54.9 mbar) for 30 min on a rotary evaporator at 37°C. Residual solvent was eliminated by further drying in a vacuum chamber for at least 1 hr. The resulting lipid films were hydrated with 2 mL HK buffer (50 mM HEPES-KOH, 120 mM K-acetate, pH 7.4) and vigorously vortexed with glass beads. The resulting multilamellar vesicles suspension was subjected to five freeze-thaw cycles (liquid nitrogen/37°C) followed by extrusion through a 0.2 µm polycarbonate filter using a mini-extruder (Avanti Polar Lipids). Freshly extruded liposomes prepared on the same day of the experiment or used for no more than two days were shielded from light were used for all experiments. Dynamic Light Scattering Measurements were performed using a DynaPro instrument with a Flex-99 correlator (ProteinSolutions) and quartz cuvettes. The instrument sensitivity was set to 30% with a maximum acquisition time of 10 s and a signal-to-noise threshold of 2.5. The cuvette holder temperature was maintained at 25°C. The buffer refractive index was set to 1.333. For kinetics experiments, 20 min autocorrelation curves were acquired from 200 nm extruded liposomes (50 μM lipid concentration) in degassed HK buffer (50 mM HEPES-KOH, 120 mM K-acetate, pH 7.4) with 1 μM of StarLok1 in acetate buffer added after 1 min. Alternatively, StarLok2 and StarLok3 were tested. Data filtering and analysis were performed using DYNAMICS software (v.5.25.44) with a maximum Sum Of Square (SOS) limit at 2100, under baseline limit at 0.995, over baseline limit at 1.02, under amplitude limit of 0.05. For the correlation function, the first 2 coefficients were ignored, and the analysis was truncated at channel 120. The autocorrelation functions were then analyzed using the regularization algorithm. Lipid transfer assay NBD-lipid transfer was measured as previously described 59 with modifications. Fluorescence was recorded using a Cary Eclipse Fluorescence Spectrophotometer (Agilent) equipped with a temperature controller set to 30°C. In a 500 µL quartz SUPRASIL rectangular cuvette (PerkinElmer), stirred with a magnetic bar, 200 µM donor liposomes (L A ) containing 2 mol% Rhod-PE and 5 mol% NBD-labeled lipid (NBD-PC, NBD-PE, NBD-PS, or NBD-ceramide) were suspended in HK buffer (50 mM Hepes, 120 mM K-acetate, pH 7.4) pre-warmed at 30°C. NBD fluorescence was monitored at 538 nm (emission slit = 5 nm) with excitation at 473 nm (slit = 2.5 nm), photomultiplier voltage of 800 V, and acquisition interval of 0.5 s. At t = 1 min, 200 µM acceptor liposomes (L B ; DOPC only) were added. After 3 min of slow spontaneous transfer, StarLok1 (1.60 µL; 5 µM final concentration) was injected, resulting in a significant increase in fluorescence indicative of NBD-lipid transfer from L A to L B . As a negative control, 1.73 µL of proteinase K-digested StarLok1 was tested. Digested samples were prepared by incubating StarLok1 with Proteinase K (20 mg/mL, RNA grade; Invitrogen) at a 1:12 (v/v) ratio for 1 h at 42°C, followed by heat inactivation for 5 min at 98°C. Complete proteolysis was confirmed by SDS-PAGE analysis. The amount of NBD-lipid transferred was quantified by measuring the loss of FRET between NBD and Rhod-PE, normalized as: Conc = 5 × ((F – F min ) / (F max – F min )) where F is the recorded fluorescence, F max is the mean fluorescence over 30 s prior to protein injection, and F min is the mean fluorescence at equilibrium (t = 12 min, averaged over 3 min) determined in an independent experiment without protein using pre-equilibrated liposome populations L A (2 mol% Rhod-PE) and L B , both containing 2.5 mol% NBD-lipid. The coefficient, 5 reflects the 5 µM NBD-lipid accessible in the external leaflet of L B . Baseline correction was performed by subtracting the mean normalized signal from control experiments (n = 3) in which 1.64 µL of vehicle buffer (PBS containing 1% N-lauroylsarcosine) was injected instead of protein; no increase in lipid transfer was observed under these conditions. Initial transfer rates (Vᵢ ± SEM) were determined from the slope of a linear fit (least-squares regression, unweighted) to data collected over 3 s post-injection, treating each replicate as an individual data point. Statistical analysis was performed using two-way ANOVA with Tukey’s post-hoc test. All analyses were conducted in GraphPad Prism (v10.2.3). Yeast expression E. coli codon optimized sequences for StarLok1, StarLok2, StarLok3 and StarHod1 were cloned into the yeast integration vector pCD256 under a TEF1 promoter and fused with a C-terminal yeGFP. The expression construct was integrated into the URA3 locus of background strain W303a after double digestion of pCD256 with Pme1/Pac1 and chemical transformation. Clones were isolated by selective plating with G418 and grown in CSM medium with 2.0% w/v glucose. The C2 domain of Bovin Lactadherin N-terminally fused to mRFP was a gifted from Sergio Grinstein (Addgene plasmid #74061) was cloned into a pRS415 vector under the control of a PGK1 promoter and terminator sequences. Yeast cells expressing StarLok1 were transformed and selected on CSM-Leu with 250 µg/mL G418, then grown in CSM-Leu medium with 2.0% w/v glucose. For imaging, stationary phase cells were added to concanavalin-1A-coated 8-well coverslip dishes and imaged with a Zeiss LSM 880 confocal laser scanning microscope equipped with an AxioObserver stand and a Plan-Apochromat 63×/1.4 NA Oil DIC M27 objective. Images were acquired with the following settings: GFP was excited using a 488 nm argon laser at 2.3% power, and fluorescence emission was collected in the 494.95-565.07 nm range using a photomultiplier tube (PMT) detector set at 750 V. Brightfield (BF) images were simultaneously captured using a non-descanned transmission PMT detector. Imaging parameters included a pixel dwell time of 4.1 μs, 8× line averaging, and a confocal pinhole of 0.997 Airy units (50.4 μm) to optimize the signal-to-noise ratio while maintaining optical sectioning. Images were digitized as 16-bit single optical sections with a frame size of 1024×1024 pixels and subsequently cropped to 512×512 pixels for further processing. The physical pixel size was 65.9 nm, yielding a field of view of 33.7×33.7 μm. For super-resolution imaging, the same microscope was used with its Airyscan detector. Z-stacks were collected with a step size of 0.18 μm, covering a total depth of 11.88 μm (66 slices). For optimal resolution, the pixel size was set to 0.04×0.04 μm in the x-y dimension, yielding voxel dimensions of 0.04×0.04×0.18 μm. For dual-channel imaging (FFP/mRFP), Z-stacks were collected with 0.185 μm steps (75 slices, 13.87 μm total depth) at 0.0488×0.0488 μm pixel size, yielding voxel dimensions of 0.0488×0.0488×0.185 μm. Excitation was performed using 488 nm (argon laser) and 561 nm (DPSS laser) at 2% power each. Raw Airyscan images were processed using the ZEISS ZEN software with the standard Airyscan processing algorithm in 3D mode. In FIJI (imageJ), the “Sharpen” filter was applied once to enhance structural details. No additional processing was performed on individual slices to maintain the integrity of the original signal distribution. For visualization purposes, all images were cropped to 512×512 pixels to focus on the regions of interest. Isolation of GFP-tagged Asgard archaeal proteins for lipidomic analysis StarLok1, StarLok2, StarLok3, and StarHod1 GFP-fusion proteins were expressed in S. cerevisiae W303 grown in 50 mL YPD medium at 30°C for 24 h, following 1:100 inoculation from an overnight culture. Cells were harvested by centrifugation (3,000 ×g, 5 min, 4°C), washed once with ice-cold sterile water, and resuspended in 500 µL lysis buffer (10 mM Tris-HCl, 150 mM KCl, 1 mM MgCl₂, pH 7.2; filtered and degassed) supplemented with 1 mM DTT, Complete EDTA-free protease inhibitor cocktail (1 tablet per 20 mL; Roche), and PhosSTOP phosphatase inhibitor (2 tablets per 20 mL; Roche). Cell lysis was performed by cryogenic grinding. The cell slurry was transferred to a ceramic mortar pre-chilled and maintained at −196°C with liquid nitrogen and ground to a fine powder with a pestle. The frozen powder was transferred to a 2 mL microcentrifuge tube and thawed on ice. To reduce viscosity, samples were supplemented with 50 µL DNase I solution (20 µg/mL DNase I Grade II from bovine pancreas [Roche] in 5 mM MgCl₂). Lysates were then clarified by centrifugation (20,000 ×g, 15 min, 4°C), yielding approximately 1 mL of supernatant. For affinity purification, clarified lysate was transferred to a 2 mL round-bottom tube containing 50 µL GFP-Trap Magnetic Particles (M-270; Chromotek), pre-washed and equilibrated in a wash buffer (10 mM Tris-HCl, 150 mM KCl, 1 mM MgCl₂, pH 7.2; filtered and degassed) according to the manufacturer’s instructions. The suspension was incubated for 1 h at 4°C with end-over-end rotation. Beads were washed five times with the wash buffer and stored at 4°C until lipid extraction. Analysis of lipids bound to GFP-StarAsgard proteins Total lipids bound to GFP-Star Asgard protein were extracted via an adapted BUME method 97 . Magnetic particles bound to GFP-StarAsgard proteins were resuspended in 500 µL of 3:1 (v/v) butanol:methanol and vortexed until complete resuspension. The suspension was transferred to a 12×75 mm conical glass tube, combined with 500 µL of 3:1 (v/v) heptane:ethyl acetate and 500 µL of 1% acetic acid, and centrifuged for 5 min at 3,100 ×g. The upper phase (450 µL) was transferred to an amber glass vial with a PTFE rubber-lined cap (Wheaton) and evaporated at 40°C for 1 h using [evaporation method]. The dried lipid extract was resuspended in 50 µL of 18:1:1 (v/v/v) 2-propanol:dichloromethane:methanol and vortexed for 3 min. Samples were centrifuged for 1 min at 3,100 ×g and stored at −20°C until analysis For lipidomics analysis, deuterated internal standard (Avanti) representing each of the 15 lipid classes analyzed were spiked into the resuspended samples. These allow for semi-quantification of species within these classes. Other potentially present lipid species, such as ceramide derivatives, glycosylated lipids, and non-sterol terpenes, were not quantified using this method. Polar lipids were measured according to a previously described method 98 , with slight modifications. The Vanquish UHPLC (Thermo Fisher Scientific) was interfaced with a QExactive mass spectrometer (Thermo Fisher Scientific). A Waters T3 1.6 µM 2.1 mm x 150 mm column was used for chromatographic separation using a step gradient from 25% buffer A (10 mM ammonium formate and 0.1% formic acid in water) to 100% buffer B (70/30 isopropanol/acetonitrile with 10 mM ammonium formate and 0.1% formic acid) over 35 min. Flow rate was set at 0.3 mL/min. This LC gradient minimizes ion suppression. Lipid samples were analyzed using data-dependent acquisition (DDA) with an NCE of 30 in negative mode and an NCE of 25 in positive mode. Ion source parameters were: Sheath Gas 48 AU; Aux Gas 11 AU; Sweet Gas 1 AU; Spray Voltage 3.5 kV for positive mode and 2.5 kV for negative mode; Capillary Temp. 250°C; S-lens RF level 60 AU; Aux Gas Heater Temp. 413°C. All ions in the mass range of 200-1200 m/z were monitored. MS1 resolution was set at 70,000 (FWHM at m/z 200) with an automatic gain control (AGC) target of 1e6 and Maximum IT of 200 ms. MS2 resolution was set to 17,500 with an AGC target of 5e4, fixed first mass of 80 m/z, and Maximum IT of 50 ms. The isolation width was set at 1.2; Dynamic Exclusion was set at 3 s. Lipids were identified and quantified with Lipid Data Analyzer Software. Interpro Sequence alignments and phylogeny of START proteins in Asgards The initial dataset consists of the 56 identified sequences corresponding to the InterPro accession number IPR023393 in annotated Asgard strains including Lokiarchaeia (MK-D1, CR_4, b31, b32 and GC14_75), Thorarchaeia (SMTZ1_45, SMTZ1_83 and AB25), Heimdallarchaeia (including LC_3 and B3_JM_08 both reclassified as Hodarchaeales, LC_2 as Kariarchaeaceae, and AB_125 as Heimallarchaeiaceae 25 , 26 ) and Helarchaeales. One sequence (A0A135VUP7) from Thor was removed because it was identical to A0A135VGP2. Full length protein sequences were aligned using MAFFT L-INS-i with default parameters and Blosum62 matrix 99 . The alignment results was used to compute a ML phylogeny using PhyML server (PhyML 3.3.2), with Smart Model Selection based on Bayesian information Criteria (SMS BIC), using a neighbor joining starting tree (BioNJ) 100 . The inference was then reiterated in IQTree 101 , using the same substitution model, frequency parameters, and seed, and branch support was assessed using SH-aLRT (1000 replicates and ultrafast bootstrap (UFBoot2, 1000 replicates). The tree was then displayed and annotated using the iTOL online tool 102 and a singular sequence from Helarchaeales was selected as a root. NCBI sequences alignment and phylogeny of START proteins in Asgards Asgard proteome sequences were retrieved from the NCBI database (GenBank Release 264), comprising 711,493 Identical Protein Groups (IPGs). Protein domain analysis was performed using InterProScan version 5.72-103.0 103 deployed via Docker Desktop 4.37.2 (179585) on macOS. Analysis was restricted to Gene3D, Pfam, and SUPERFAMILY search programs. A custom bash script divided protein sequences into smaller batches and ran analyses in parallel (two concurrent processes, two CPU cores each). The pre-calculated match lookup service was utilized with default configuration. Output files were organized by batch and the 508,901 newly annotated sequences were concatenated for downstream analysis. After assigning taxonomic ranks to each sequence using NCBI annotation, 411 START domains were identified using InterPro annotations. (SSF55961; G3DSA:3.30.530.20; PF10604; PF11485; PF08982; PF06240; PF19569; PF02121; PF08327; PF03364; PF01852; PF00407; IPR023393). Each sequence was trimmed according to the longest non-overlapping domain coordinate given by the InterPro Analysis. A subset of START domain-containing proteins in the Thorarchaeia clade contained two domains; these were retained as distinct protein sequences. The trimmed sequences were aligned using Muscle5 104 . ML phylogenies were inferred with IQ-Tree following model selection using ModelFinder. Branch support was assessed using SH-aLRT (1000 replicates and ultrafast bootstrap (UFBoot2, 1000 replicates). Five monophyletic domain classes were identified.. To represent domain abundance, the average number of species in each Asgard archaeal taxon was estimated using seven universal single-copy proteins (SecY, uS13, uS3, uS4, aIF2, Arginine and Serine RNA ligase) among the 508,901 annotated sequences. The mean count of these marker proteins per taxon was used as a proxy for species abundance. START domain abundance in each taxon was normalized by the estimated species abundance. Large sequence dataset construction A set of 83 species of archaea including 14 Asgards, 4 Stygia, 4 Acherontia, 18 Stenoarchaea, 5 Methamonada, 4 Diaforarchaea, 31 TACK group members and 2 DPANN group members, was collected based on recent phylogeny 105 . Similarly, 32 eukaryotes were selected including Obazoa, Amoebozoa, Excavata, Cryptista, Archaeplastida and TSAR reflecting previous molecular-clock analysis 106 . 46 Bacterial species were also selected a posteriori, to increase the depth of the tree by including members of Acidobacteriota, Bdellovibrionota, Campylobacterota, Chrysiogenota, Coprothermobacterota, Deferribacterota, FCB group, Fusobacteriota, Myxococcota, Pseudomonadota (Alpha, Beta and Gammaproteobacteria), PVC group, Terrabacteria group, (Actinomycetota, Deinococcota), Thermodesulfobacteriota and Spirochaetota. For each species, START-containing protein sequences are collected using IPR023393 accession as a request to Uniprot and then used CD-HIT (-c 0.9 -n 5) to identify putative in-paralog groups 107 . For each of these groups, spurious redundant sequences were manually removed depending on their annotation in genomic databases such as genbank, Gramene, Flybase, EmsemblMetazoa and RefSeq. When locus annotations were lacking between every in-paralog, the longest transcript was selected. From this new protein dataset, as a list of accession file, ProtDomRetriever.py was executed using as an input the InterPro entries IPR023393, CATH G3DSA:3.30.530.20, and SUPERFAMILY SSF55961. Reduced sequence-based Maximum Likelihood phylogeny of START domains A curated pool of 842 START domains derived from 774 protein sequences was assembled using ProtDomRetriever.py. From this pool, specific sequence subsets were defined to generate the phylogenetic trees presented in the main and supplementary figures, enabling comparisons across START clades or within major domain groups. The sequences were aligned using MAFFT (v7) 108 . To empirically determine optimal alignment parameters, we developed MAFFT ScoreNGo.py, a wrapper that systematically evaluates alternative MAFFT configurations. The following alignment strategies were tested: G-INS-i, E-INS-i, and L-INS-i, using both BLOSUM62 and BLOSUM80 scoring matrices, together with a range of gap-opening, gap-extension, and local alignment penalty parameters. In total, 337 parameter combinations were evaluated using the standard search mode. Each resulting alignment was scored using a conservation-based metric implementing Jalview’s per-column conservation scheme 109 , derived from the AMAS method 110 . For each alignment column, conservation was evaluated based on agreement across 10 physicochemical property classes, with columns exceeding a defined gap threshold excluded from scoring. Per-column conservation scores (0–11) were summed to obtain a global conservation score for each alignment. For each dataset, the alignment yielding the highest global conservation score was retained for downstream phylogenetic inference. This empirical optimization strategy was designed to maximize residue-property consistency across the conserved START domain core, improving alignment coherence and preserving phylogenetically informative sites. Alternative alignments generated with MUSCLE5 were also evaluated using the conservation-based scoring framework described above. In direct comparisons on selected START domain datasets, MUSCLE5 produced lower global conservation scores and more gap-dense alignments relative to MAFFT under optimized parameters, reducing coherence across conserved core residues. MAFFT was therefore used for phylogenetic inference in the tree-of-life START domain analyses. ML phylogenies were inferred using IQ-TREE with extended model selection implemented via ModelFinder. ModelFinder was used to evaluate a comprehensive set of substitution models, including mixture models (C10, C20, C40, C60), LG4M and LG4X models, alternative equilibrium frequency schemes (F, FO, FQ), nuclear substitution matrices, and multiple rate heterogeneity models (E, I, G, I+G, R, I+R; 4–20 categories). The best-fitting model was selected according to the Bayesian Information Criterion (BIC) for each dataset. Branch support was assessed using SH-aLRT (1,000 replicates) and ultrafast bootstrap (UFBoot2, 1,000 replicates). The trees were displayed and annotated with iTol 102 . Large structural phylogeny of START domains AlphaFold structures were retrieved from Alphafold Protein Structure database. A trimming algorithm was applied to the PDB structures using the position file generated by ProtDomRetriever, to generate new .pdb files excluding any atoms that are not part of the START domain. When multiple START domains are present in a structure, each of these domains are extracted and copied in an independent new file. This set of trimmed structures was submitted to the FoldTree (build d0f0239) pipeline 60 , using Google Colab Notebook on a Python3 runtime accelerated by a A100 GPU. The rooted tree was then displayed and annotated with iTol without modifying the topology. From this tree, a smaller collapsed cladogram was constructed by deleting all sequences not belonging to a known clade of functional protein. Taxonomic congruence was evaluated by comparing the START domain phylogeny with a synthetic species tree extracted from the Open Tree of Life (OTOL) database 111 using the induced_subtree API. After pruning to identical taxon sets, topological agreement among major eukaryotic clades and lineage-specific paralogous expansions was assessed using a tanglegram representation. Structural comparison analysis Structural comparisons were performed using Foldseek (exhaustive search mode) to compute all-versus-all TM-scores across 805 trimmed AlphaFold2 structural models. A pairwise distance matrix (1 − TM-score) was constructed and reduced to two dimensions by t-SNE following PCA pre-processing (50 components), using scikit-learn. Ligand-binding cavity volumes were derived from cavity volumes previously computed using CavitOmiX. Figures were generated using Plotly. Schematic Proposed relationships between START proteins in the prokaryotic ancestors of eukaryotic cells (left) and their localization in eukaryotes. StarAsg1 is a putative membrane-bound lipid binding protein in Asgards, which could share a common ancestry with several START domain proteins acting as LTPs. These are predominantly localized to membrane contact sites between organelles. StarAsg2 and StarAsg3, proteins of unknown functions in Asgards, share an origin with the conserved HSP90 co-chaperone in the cytosol of eukaryotes. RatA, a START domain protein in alpha-proteobacteria, shares ancestry with the mitochondrial protein COQ10. Download figure Open in new tab Acknowledgments Yongxuan Su and the UCSD Molecular Mass Spectrometry Facility aided in analyses. Deshmukh, Devaraj, Ghosh and Herzik labs provided instrumentation support. Paula Welander and Buzz Baum provided helpful discussions. The work was supported by the National Institutes of Health (GM142960), the Moore–Simons Project on the Origin of the Eukaryotic Cell (GBMF9734, grant DOI 10.37807/GBMF9734), and the Paul G. Allen Family Foundation. Funder Information Declared National Institutes of Health , R35-GM142960 Gordon and Betty Moore Foundation, https://ror.org/006wxqw41 , GBMF9734 Paul G. 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Share A phospholipid transporter in Asgard archaea sheds light on the origin of eukaryotic lipid transfer proteins Nicolas-Frédéric Lipp , Elida Kocharian , Itay Budin bioRxiv 2025.05.16.653879; doi: https://doi.org/10.1101/2025.05.16.653879 Share This Article: Copy Citation Tools A phospholipid transporter in Asgard archaea sheds light on the origin of eukaryotic lipid transfer proteins Nicolas-Frédéric Lipp , Elida Kocharian , Itay Budin bioRxiv 2025.05.16.653879; doi: https://doi.org/10.1101/2025.05.16.653879 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Evolutionary Biology Subject Areas All Articles Animal Behavior and Cognition (7635) Biochemistry (17691) Bioengineering (13892) Bioinformatics (41937) Biophysics (21452) Cancer Biology (18588) Cell Biology (25504) Clinical Trials (138) Developmental Biology (13378) Ecology (19899) Epidemiology (2067) Evolutionary Biology (24320) Genetics (15609) Genomics (22506) Immunology (17736) Microbiology (40394) Molecular Biology (17181) Neuroscience (88605) Paleontology (666) Pathology (2832) Pharmacology and Toxicology (4824) Physiology (7641) Plant Biology (15156) Scientific Communication and Education (2045) Synthetic Biology (4294) Systems Biology (9825) Zoology (2271)