Most eukaryotic, secreted proteins travel through the conventional Golgi-ER secretory pathway. However, some proteins are unconventionally secreted independent of this route. We have shown that the major sperm protein (MSP) domain of VAPB/ALS8 is cleaved from the rest of the protein and secreted. MSP can be detected in human blood and cerebrospinal fluid (CSF), and its levels correlate with amyotrophic lateral sclerosis (ALS) pathogenesis. The goal of this project is to understand how VAPB MSP is proteolytically processed, secreted, and regulated. VAPB is a ubiquitously expressed type II membrane protein found anchored into the endoplasmic reticulum with the N-terminal MSP domain extending into the cytosol. The secreted MSP domain does not harbor a signal peptide characteristic of conventionally secreted proteins. C. elegans null mutants for vpr-1, the Vapb homolog, are sterile and have striated muscle mitochondrial abnormalities. Results from our lab provide evidence that the nervous system, germ line, and intestine are cellular sites of VPR-1 MSP secretion. Based on studies of C. elegans sperm MSPs, we hypothesize that VAPB MSP is secreted in a regulated fashion via an unconventional mechanism. In order to test our hypothesis, we developed an RNAi screening method using transgenic C. elegans to identify genes required for MSP secretion. vpr-1(tm1411) null mutants expressing VPR-1 only in the intestine (pGES-1::vpr-1) are fertile and can be grown as transgenic homozygotes, presumably because the MSP is secreted into the pseudocoelom. We predicted that RNAi of genes essential for VPR-1 MSP secretion would cause sterility. Using expression data, I have selected a set of 420 genes for a pilot RNAi screen. I have currently screened 210 RNAi clones and identified 10 candidate clones that cause severely reduced fertility in transgenic vpr-1 mutants, but not in wild-type worms. Genome-editing techniques will be used to further characterize the role of selected candidates. I am also conducting a structure/function analysis to identify regulatory regions within VPR-1 necessary for MSP secretion. Finally, we have been characterizing multiple mouse Vapb mutants with skeletal muscle mitochondrial defects, in collaboration with Jin Chen’s lab at Vanderbilt. I plan to use western blots of isolated mouse tissues to help define the mammalian cell types that proteolytically process VAPB to liberate MSP. VAPB MSP injections or drugs that improve VAPB MSP secretion could be therapeutic for ALS patients.