Genomic screen for vacuolar protein sorting genes in Saccharomyces cerevisiae

Abstract

The biosynthetic sorting of hydrolases to the yeast vacuole involves transport along two distinct routes referred to as the carboxypeptidase Y and alkaline phosphatase pathways. To identify genes involved in sorting to the vacuole, we conducted a genome-wide screen of 4653 homozygous diploid gene deletion strains of Saccharomyces cerevisiae for missorting of carboxypeptidase Y. We identified 146 mutant strains that secreted strong-to-moderate levels of carboxypeptidase Y. Of these, only 53 of the corresponding genes had been previously implicated in vacuolar protein sorting, whereas the remaining 93 had either been identified in screens for other cellular processes or were only known as hypothetical open reading frames. Among these 93 were genes encoding: 1) the Ras-like GTP-binding proteins Arl1p and Arl3p, 2) actin-related proteins such as Arp5p and Arp6p, 3) the monensin and brefeldin A hypersensitivity proteins Mon1p and Mon2p, and 4) 15 novel proteins designated Vps61p-Vps75p. Most of the novel gene products were involved only in the carboxypeptidase Y pathway, whereas a few, including Mon1p, Mon2p, Vps61p, and Vps67p, appeared to be involved in both the carboxypeptidase Y and alkaline phosphatase pathways. Mutants lacking some of the novel gene products, including Arp5p, Arp6p, Vps64p, and Vps67p, were severely defective in secretion of mature alpha-factor. Others, such as Vps61p, Vps64p, and Vps67p, displayed defects in the actin cytoskeleton at 30 degrees C. The identification and phenotypic characterization of these novel mutants provide new insights into the mechanisms of vacuolar protein sorting, most notably the probable involvement of the actin cytoskeleton in this process.

Figure 1

Identification of CPY-secreting strains. The collection of 4653 homozygous diploid deletion strains developed by the Saccharomyces Genome Deletion Project was screened using a CPY colony blot assay (Roberts et al., 1991). The membranes were subjected to immunoblotting with mouse anti-CPY at 1:1000 dilution. Results for either one 96-well plate (a) or examples of several mutants taken from different blots (b) are shown. Wild-type (wt) and vps39 strains are included for comparison.

Figure 2

Analysis of CPY, PrA, and ALP processing. Wild-type (wt) and deletion strains were pulse-labeled with [³⁵S]methionine for 10 min (first lane of each pair) and chased for 10 min (second lane of each pair). Sequential immunoprecipitations were performed from the lysates by using antibodies against CPY (a), PrA (b), or ALP (c) as described in MATERIALS AND METHODS. Results are shown for wild-type (wt) and 24 mutant strains. The positions of precursor (p) and mature (m) forms of the hydrolases examined are indicated. The p1 and p2 precursor forms of CPY were not well resolved for all strains, in some cases due to aberrant Golgi glycosylation that resulted in comigration of both precursor species. A summary of results for these and other strains not shown in this figure is presented in Table 3.

Figure 3

Visualization of vacuole morphology in wild-type and mutant strains by light microscopy. Vacuole morphology was examined by labeling with FM4-64 as described in MATERIALS AND METHODS. The photographs were taken with both fluorescence and a low level of transmitted light. Results are shown for wild-type (wt), vps39 (as a control for aberrant vacuole morphology), and 23 selected vacuolar sorting mutants identified in our screen. Many of the deletions resulted in fragmented vacuole morphology, which was intermediate, and not as severe as the class B vps mutants (vps39). These include arf1, arl1, arl3, mon1, mon2, mdm20, tpm1, dor1, cod2, cod3, vps61, and vps67. Bar, 9 μm.

Figure 4

Visualization of the actin cytoskeleton in wild-type and mutant strains by light microscopy. The actin cytoskeleton was examined by labeling fixed log-phase cells with 1.1 μM Oregon green-phalloidin at 30°C for ∼16–20 h as described in MATERIALS AND METHODS. Results are shown for wild-type (wt), vps39, and 23 selected vacuolar sorting mutants. The most common phenotype observed was the decrease in number or an absence of polarized actin cable in the mother cells, as is seen in arf1, mdm20, tpm1, and vps61. Other phenotypes included reduced polarized actin cables with the appearance of a large actin aggregate in the mother cell, seen in vps36 and vps65 (arrowheads), and slightly disorganized actin filaments, observed in arp5 and arp6. Bar, 9 μm.

Figure 5

Analysis of α-factor secretion and processing. (a) To test for α-factor secretion, wild-type and mutant strains were grown to log phase and serially diluted to the concentrations indicated, and equal amounts were spotted on YEPD plates containing a freshly spread lawn of the α-factor–sensitive strain RC634 (MAT a, sst1-3), as described in MATERIALS AND METHODS. The plates were then incubated at 30°C for 48 h to assess α-factor secretion as indicated by the relative growth inhibition of the sst1-3 mutant strain (halo). (b) To test for processing of α-factor, strains were metabolically labeled with [³⁵S]methionine at 25°C for 7.5 min as described in MATERIALS AND METHODS. Mature α-factor (mαf), α-factor–processing intermediates and pro-α-factor were immunoprecipitated with anti-α-factor antibodies, and resolved by SDS-PAGE (4–20% acrylamide gradient gels). Various forms of α-factor were detected, including pro-α-factor (pαf) that includes a range of high molecular weight intermediates containing the proregion and various degrees Golgi-derived glycosylation (∼ 90– 150 kDa), unglycosylated core-α-factor (unglyc) (∼20 kDa), ER core-glycosylated α-factor (core) (∼26 kDa), low molecular weight intermediates in α-factor processing (interαf) that most likely represent the tetrapeptide in various states of processing with the proregion removed (∼6–8 kDa), and the mature α-factor (mαf), the 13-amino acid terminally processed secreted form (∼2 kDa).