Research in this laboratory centers around the activities and genesis of the lysosome-like vacuole of the yeast Saccharomyces cerevisiae. Yeast is amenable to genetic, biochemical, molecular biological and cell biological analysis; the sequence of its genome is known.

Human diseases caused by multi-organellar disorders.

Proteins are delivered to the yeast vacuole by three different cellular pathways. The pathway that is best known and most thoroughly investigated is a branch of the secretory pathway. The endosomal pathway begins with budding off of vesicles from the late Golgi, which are delivered to the prevacuolar compartment (a late endosome, apparently), where they fuse through the agency of Pep12p, a syntaxin homolog, the SNARE complex, and ancillary proteins. Proteins move from there to the vacuole. The endosomal pathway is followed by the inactive precursors to the vacuolar hydrolases Carboxypeptidase Y (CpY), Carboxypeptidase S (CpS), Protease B (PrB), Protease A (PrA), and an integral membrane subunit of the vacuolar ATPase (Vph1p). A direct route from the late Golgi to the vacuole exists; it is followed by the inactive precursor to Alkaline phosphatase (ALP) and by the vacuolar syntaxin, Vam3p/Pth1p. The direct route bypasses the endosome and is dependent on the AP3 adaptin complex. There is also a route directly from the cytoplasm to the vacuole; the inactive precursor to Aminopeptidase I (ApI) follows this route.

More than 50 genes are required for biogenesis of the vacuole and delivery of proteins to it. We have been studying several of these genes: PEP3, PEP5, PEP7, PEP8, PEP11, PEP12, PTH1, and VPS8. Pep12p, a syntaxin homologue, is cytoplasmically oriented and inserted C-terminally in the endosomal membrane, where it is thought to participate in vesicular fusion. It interacts with Vps45p, a Sec1p homologue, and with Pep7p. The rab5 homologue Vps21p also is required at this step. VAM3/ PTH1 was identified by sequence homology to PEP12. Disruption leads to a defect in delivery of proteins via all three routes to the vacuole: the direct and endosomal routes from the Golgi and the cytoplasmic pathway.

Genetic evidence suggested that Pep5p might be required for Golgi to endosome as well as for the end steps of the cytoplasmic, and direct and endosomal routes from the Golgi to the vacuole. Recently we have used temperature sensitive mutants of PEP3 and PEP5 to demonstrate that Pep3p and Pep5p are required for traffic from the Golgi to the endosome, for recycling from the endosome to the Golgi, for delivery of hydrolase precursors from all three pathways at the vacuole (the terminal steps in the three pathways). They are also required for at least one step in the endocytotic pathway after internalization and before the late endosome.   In recent experiments we have shown that Vps33p, a Sec1p homologue, is required in all reactions in which Pep3p and Pep5p participate.   Vps33p interacts with Pep12p, the endosomal syntaxin, as does Sec45p.   Both Vps33p and Vps45p are Sec1p homologues.   This is the first report that two different Sec1p homologues participate in the same trafficking step, namely, fusion at the endosome. Other investigators have shown that Pep3p, Pep5p, Vps16p, and Vps33p act together in a complex at the vacuolar membrane.   Future work should reveal the ancillary proteins that are step-specific.

Pep3p/Pep5p functions in multiple routes of protein transport to the vacuole.

We have cloned the structural genes for three of the vacuolar hydrolases. The pro region of the protease B precursor functions as an intramolecular chaperone. We have isolated mutations in the pro-encoding region that cause a Prb- phenotype. A novel protein, Pbn1p, was identified whose function is necessary if Prb1p is to fold properly to allow the autocatalytic scission of the propeptide from Prb1p in the endoplasmic reticulum. In the pbn1 mutant, misfolded Prb1p is exported from the endoplasmic reticulum and degraded. PBN1 is an essential gene. We hope to recover conditional lethal pbn1 mutants so that we can identify the essential function. Pbn1p was shown to interact with the pro region of the PrB precursor by two-hybrid analysis. As the pro region and Pbn1p have short regions of helix that are predicted to form helical coiled coils and because deletion of this region from the PrB precursor abrogates the two-hybrid interaction and prevents maturation of the precursor, we are mutating the heptad repeat in the two polypeptides to investigate the basis of the interaction.

Subramanian S, Woolford CA, Jones EW. The Sec1/Munc18 protein, Vps33p, functions at the endosome and the vacuole of Saccharomyces cerevisiae. Molecular Biology of the Cell 2004. Published March 26 2004 as 10.1091/mbc E03-10-0767.

Chang HJ, Jones EW, Henry SA. Role of the unfolded protein response pathway in regulation of INo1 and in the sec114 bypass mechanism in Saccharomyces cerevisiae. Genetics 2002; 162:29-43.

Srivastava A, Woolford CA, Jones EW. Pep3p/Pep5p complex: A putative docking factor at multiple steps of vesicular transport to the vacuole of Saccharomyces cerevisiae. Genetics 2000; 156: 105-122.

Woolford CA, Bounoutas G, Frew S, Jones EW. Genetic interaction with vps8-200 allows partial suppression of the vestigial vacuole phenotype caused by a pep5 mutation in Saccharomyces cerevisiae. Genetics 1998; 148: 71-83. PMID: 9475722.

Naik R, Jones EW. The PBN1 gene of Saccharomyces cerevisiae: An essential gene that is required for post-translational processing of the Proteinase B precursor. Genetics 1998; 149: 1277-1292. PMID: 9649520.