Murine astrotactins 1 and 2 have similar membrane topology and mature via endoproteolytic cleavage catalyzed by signal peptidase

Astrotactins 1 (Astn1) and Astn2 are membrane proteins that function in glial-guided migration, receptor trafficking and synaptic plasticity in the brain, as well as in planar polarity pathways in skin. Here, we used glycosylation mapping and protease-protection approaches to map the topologies of mouse Astn1 and Astn2 in rough microsomal membranes (RMs), and found that Astn2 has a cleaved N-terminal signal peptide (SP), an N-terminal domain located in the lumen of the RMs (topologically equivalent to the extracellular surface in cells), two transmembrane helices (TMHs), and a large C-terminal lumenal domain. We also found that Astn1 has the same topology as Astn2, but we did not observe any evidence of SP cleavage in Astn1. Both Astn1 and Astn2 mature through endoproteolytic cleavage in the second TMH; importantly, we identified the endoprotease responsible for the maturation of Astn1 and Astn2 as the endoplasmic reticulum signal peptidase. Differences in the degree of Astn1 and Astn2 maturation possibly contribute to the higher levels of the C-terminal domain of Astn1 detected on neuronal membranes of the central nervous system. These differences may also explain the distinct cellular functions of Astn1 and Astn2, such as in membrane adhesion, receptor trafficking, and planar polarity signaling.