Quantitative proteomics of yeast post-Golgi vesicles reveals a discriminating role for Sro7p in protein secretion

Abstract

We here report the first comparative proteomics of purified yeast post-Golgi vesicles (PGVs). Vesicle samples isolated from PGV-accumulating sec6-4 mutants were treated with isobaric tags (iTRAQ) for subsequent quantitative tandem mass spectrometric analysis of protein content. After background subtraction, a total of 66 vesicle-associated proteins were identified, including known or assumed vesicle residents as well as a fraction not previously known to be PGV associated. Vesicles isolated from cells lacking the polarity protein Sro7p contained essentially the same catalogue of proteins but showed a reduced content of a subset of cargo proteins, in agreement with a previously shown selective role for Sro7p in cargo sorting.

Figure 1. Distribution of the post-Golgi vesicle marker Snc1/2p and Sso1/2 in sorbitol vesicle gradient fractions from sec6-4 and sec23-1 strains

A) Cells were cultured and lysates prepared and fractioned as described in Materials and Methods. Samples from each fraction of the gradient were heated in sample buffer at 95°C for 5 min, and resolved by SDS–PAGE on 12.5% Tris–HCl gels (Biorad). Separated proteins were transferred for western blot with antiserum against Sso1/2p and Snc1/2p. B) Distribution profile of Snc1/2p in the vesicle gradient fractions from sec6-4 and sec23-1 strains. The three peak fractions from the sec6-4 gradient were used for subsequent analysis. For the mock strain (sec23-1), fractions corresponding to the sec6-4 peak fractions were used. C) Relative strength of Snc1/2p signals from peak fractions 7, 8 and 9 of the sec6-4 and sec23-1 preparations.

Figure 2. Sub-cellular enrichment of vesicle proteins prepared from sec6-4 (open bars) and sro7Δ (filled bars) strains

Vesicle proteins were defined as proteins >2.5-fold more abundant in sec6-4 or sro7Δ fractions relative to the mock (sec23-1) strain. All sub-cellular compartment annotations were significantly enriched relative to the random expectation according to Fisher’s exact test (Bonferroni correction, p < 0.1) for either sec6-4 or sro7Δ. Localization annotations were from the Gene Ontology classification system as represented in the SGD yeast GO slim component catalogue (http://www.yeastgenome.org/cgi-bin/GO/goSlimMapper.pl).

Figure 3. Functional classification of proteins in vesicles prepared from sec6-4 (red bars) and sro7Δ (blue bars) strains

Annotations were derived from the functional ontology classification system hosted by the MIPS Comprehensive Yeast Genome Database (http://mips.helmholtz-muenchen.de/genre/proj/yeast/Search/Catalogs/catalog.jsp). Enrichment was determined as in Figure 2. Only functional processes with at least 15 annotated members were considered in the test.

Figure 4. Top 15 proteins depleted in vesicles from cells lacking SRO7

Annotations and enrichment were determined as in Figure 2.

Figure 5. Abundance of selected vesicle proteins analyzed in Table 3

A) An equal amount of protein from purified vesicles prepared from sro7Δ and sec6-4 mutants was subjected to immunoblotting as described in Materials and Methods. Protein bands of expected sizes were quantitated and results illustrated in the bar graphs (right). B) A bar graph of log(2) transformed ratio values comparing the sro7Δ/sec6-4 ratio from immunoblot with the corresponding iTRAQ values. Samples for iTRAQ analysis and immunoblot quantification were independently prepared.

Figure 6. Localization of Stl1p-GFP in wild-type and sro7Δ cells

Fluorescence images show the distribution of Stl1p-GFP in (A) wild type and (B) sro7Δ cells. Cells carrying a pUG35 STL1-GFP plasmid were grown to early exponential phase in medium with 500 µm methionine. NaCl was added to a final concentration of 0.6 m and incubation was continued for 2 h. Synthesis of Stl1p-GFP was induced by a shift to methionine-free medium (with 0.6 m NaCl) and incubation was continued for 2 h at 37°C. C) Distribution of Stl1p-GFP in purified PGVs from wild-type and sro7Δ strains. Cells transformed with the pUG35-STL1 plasmid were harvested after a 2 h shift to 0.6 m NaCl, followed by a 2 h shift to methionine free medium of the same salinity (to allow for induction of Stl1p-GFP) and 37°C. Purified PGVs were subjected to SDS–PAGE as described in Materials and Methods and Stl1p-GFP was detected by immunoblotting with anti-GFP antibody. Bands were quantitated (bar graph) and the Stl1p-GFP protein ratio in PGVs from sro7Δ relative to wild type estimated to 0.33.