Isolation and characterization of PEP3, a gene required for vacuolar biogenesis in Saccharomyces cerevisiae.

Abstract

The Saccharomyces cerevisiae PEP3 gene was cloned from a wild-type genomic library by complementation of the carboxypeptidase Y deficiency in a pep3-12 strain. Subclone complementation results localized the PEP3 gene to a 3.8-kb DNA fragment. The DNA sequence of the fragment was determined; a 2,754-bp open reading frame predicts that the PEP3 gene product is a hydrophilic, 107-kDa protein that has no significant similarity to any known protein. The PEP3 predicted protein has a zinc finger (CX2CX13CX2C) near its C terminus that has spacing and slight sequence similarity to the adenovirus Ela zinc finger. A radiolabeled PEP3 DNA probe hybridized to an RNA transcript of 3.1 kb in extracts of log-phase and diauxic lag-phase cells. CeUls bearing pep3 deletion/disruption alleles were viable, had decreased levels of protease A, protease B, and carboxypeptidase Y antigens, had decreased repressible alkaline phosphatase activity, and contained very few normal vacuolelike organelles by fluorescence microscopy and electron microscopy but had an abundance of extremely smal vesicles that stained with carboxyfluorescein diacetate, were severely inhibited for growth at 37C, and were incapable of sporulating (as homozygotes). Fractionation of ceUs expressing a bifunctional PEP3::SUC2 fusion protein indicated that the PEP3 gene product is present at low abundance in both log-phase and stationary cels and is a vacuolar peripheral membrane protein. Sequence identity established that PEP3 and VPS18 (J. S. Robinson, T. R. Graham, and S. D. Emr, Mol. Cell. Biol. 11:5813-5824, 1991) are the same gene.The vacuole in Saccharomyces cerevisiae is a polymorphic organelle that has similarities both to vacuoles in plants and to lysosomes in animal cells. Like lysosomes, yeast vacuoles are relatively acidic organelles that contain a number of proteases and other hydrolases that have relatively broad substrate specificities. Mutants that lack particular vacuolar proteases have been useful for determining the functions of specific enzymes (23,25). One search for protease-deficient mutants unexpectedly identified 16 genes, mutations in any of which gave rise to deficiencies of two or three different vacuolar hydrolase activities (PEP genes) (21). Because pep mutations are pleiotropic, the phenotypes of the mutants provide very limited information about the primary functions of the PEP gene products. At the time the genes were identified, several general hypotheses were advanced to account for the pleiotropy of the mutations: that (i) these are regulatory mutations, (ii) the mutations cause changes in the structure of the compartments containing the enzymes, or (iii) the mutations alter components of the system that places the enzymes in the compartments (21). Thus, the PEP genes appeared to provide access to cellular "capital equipment" common to the expression of more than one vacuolar protease, but it was not clear in biochemical terms what type of equipment that might be. All three of the mechanisms mentioned above (or others not thought of)...

A Novel RING Finger Protein Complex Essential for a Late Step in Protein Transport to the Yeast Vacuole

“…The four class C vps mutants (vps18/pep3, vps16, vps11/end1/pep5, and vps33/slp1) exhibit the most severe vacuolar protein sorting and morphology defects. These mutants lack any structure resembling a normal vacuole and instead accumulate small vesicles and other aberrant membrane compartments Woolford et al, 1990;Preston et al, 1991). In addition, the class C vps mutants exhibit multiple defects consistent with the absence of vacuoles, including temperature-sensitive growth defects, osmotic sensitivity, calcium sensitivity, reduced amino acid pools, and sporulation defects Robinson et al, 1988;Dulic and Riezman, 1989;Wada et al, 1990;Woolford et al, 1990;Preston et al, 1991).…”

“…VPS16 encodes a 798-amino acid protein that lacks homology to characterized proteins or protein motifs (Horazdovsky and Emr, 1993). VPS18/PEP3 and VPS11/END1/PEP5 encode hydrophillic proteins of 918 and 1029 amino acids, respectively (Dulic and Riezman, 1989;Woolford et al, 1990;Preston et al, 1991;Robinson et al, 1991), both of which contain C-terminal RING finger zinc-binding domains. The RING finger domain is important for the biological activity of the VPS18/PEP3 gene product, because vps18 cells harboring a mutation in this domain exhibit temperature-conditional CPY sorting defects (Robinson et al, 1991).…”

The Binary Interacting Network of the Conserved Oligomeric Golgi Tethering Complex

“…TRAPPI and -II are two related tethering complex that function in endoplasmic reticulum-Golgi and intra-Golgi transport and act together with Ypt1/Rab1 (21), whereas the HOPS complex (or Class C VPS complex) consisting of Vps11, Vps16, Vps18, Vps33, Vps39, and Vps41 act together with Ypt7 and SNAREs in mediating tether and fusion at the vacuole in yeast (22). The HOPS complex has been shown to function in multiple transport steps in addition to fusion at the vacuolar membrane (23)(24). Proteins homologous to components of the TRAPPs and HOPS complexes are found in mammalian cells and could function in a similar manner in their respective tethering processes.…”

The yeast homolog of H < beta > 58, a mouse gene essential for embryogenesis, performs a role in the delivery of proteins to the vacuole.

“…In addition, diploids that were homozygous for pep8 lacked any sporulation defects. Because most of the other vacuolar biogenesis mutants isolated exhibit a defect in one of the above phenotypes, it appears from these studies on the pep8 disruptants that vacuolar biogenesis per se was not affected in these mutants (Banta et al 1990;Wada et al 1990;Woolford et al 1990;Preston et al 1991;Robinson et al 1991;Raymond et al 1992).…”

“…This is in contrast to the other vacuolar biogenesis/sorting mutants. PEP3 is a gene required for vacuolar biogenesis/sorting, and cells bearing a disruption in PEP3 have been shown to secrete large amounts of the P2 precursor of CpY (Preston et al 1991;Robinson et al 1991). To determine whether the precursor form that remained intracellular was processed to mature form in pep3A-bearing cells and compare it with the intracellular processing of pep8/t cells, we carried out a kinetic analysis on the two mutants; the results are shown in Figure 5.…”