Yeast cells deleted for the SRO7/SOP1 encoded tumor suppressor homologue show increased sensitivity to NaCl stress. On exposure to growth-inhibiting NaCl concentrations, sro7⌬ mutants display a rapid loss in viability that is associated with markers of apoptosis: accumulation of reactive oxygen species, DNA breakage, and nuclear fragmentation. Additional deletion of the yeast metacaspase gene YCA1 prevents the primary fast drop in viability and diminishes nuclear fragmentation and DNA breakage. We also observed that NaCl induced loss in viability of wild-type cells is Yca1p dependent. However, a yeast strain deleted for both SRO7 and its homologue SRO77 exhibits NaCl-induced cell death that is independent on YCA1. Likewise, sro77⌬ single mutants do not survive better after additional deletion of the YCA1 gene, and both sro77⌬ and sro77⌬yca1⌬ mutants display apoptotic characteristics when exposed to growth-inhibiting salinity, suggesting that yeast possesses Yca1p-independent pathway(s) for apoptosis-like cell death. The activity of Yca1p increases with increasing NaCl stress and sro7⌬ mutants achieve levels that are higher than in wild-type cells. However, mutants lacking SRO77 do not enhance caspase activity when subject to NaCl stress, suggesting that Sro7p and Sro77p exert opposing effects on the cellular activity of Yca1p. INTRODUCTIONThe physiological cell death process of apoptosis is a morphologically distinct form of cellular suicide (Kerr, 2002), designed to remove potentially threatening or undesired cells (Vaux and Korsmeyer, 1999;Beers and McDowell, 2001). In animal cells, apoptosis occurs in an ordered series of event and is associated with activation of the programmed cell death machinery (Wyllie et al., 1980;Joza et al., 2002). Inappropriate regulation of apoptosis is linked to the pathogenesis of many human diseases, such as AIDS, cancer, autoimmune, and neurodegenerative disorders (Uren and Vaux, 1996). The process of programmed cell death can be triggered by a vast array of stimuli, and a complex network of regulators and effectors coordinates the process (Beers and McDowell, 2001;Joza et al., 2002). At the end of almost all apoptotic scenarios, a constellation of morphological characteristics emerge, including chromatin condensation, DNA fragmentation, flipping of phosphatidylserine to the outer leaflet of the plasma membrane, and breakage of the cells to small membrane-enclosed vesicles, so called apoptotic bodies (Wyllie et al., 1980). These structural features constitute the cytological hallmarks of apoptosis.The finding that cellular suicide occurs in most, if not all, multicellular organisms raises the question on how the mechanisms of apoptosis have evolved. Do these mechanisms have a common origin and have these processes first evolved in single-celled organisms? For unicellular organisms exposed to harsh conditions, cellular suicide may serve to propagate the genome to future generations if some of the members of a population are sacrificed to promote survival of others (Engelberg-Kulk...

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.

The SRO7/SOP1 encoded tumor suppressor homologue of Saccharomyces cerevisiae is required for maintenance of ion homeostasis in cells exposed to NaCl stress. Here we show that the NaCl sensitivity of the sro7⌬ mutant is due to defective sorting of Ena1p, the main sodium pump in yeast. On exposure of sro7⌬ mutants to NaCl stress, Ena1p fails to be targeted to the cell surface, but is instead routed to the vacuole for degradation via the multivesicular endosome pathway. SRO7-deficient mutants accumulate post-Golgi vesicles at high salinity, in agreement with a previously described role for Sro7p in late exocytosis. However, Ena1p is not sorted into these post-Golgi vesicles, in contrast to what is observed for the vesicles that accumulate when exocytosis is blocked in sec6-4 mutants at high salinity. These observations imply that Sro7p has a previously unrecognized role for sorting of specific proteins into the exocytic pathway. Screening for multicopy suppressors identified RSN1, encoding a transmembrane protein of unknown function. Overexpression of RSN1 restores NaCl tolerance of sro7⌬ mutants by retargeting Ena1p to the plasma membrane. We propose a model in which blocked exocytic sorting in sro7⌬ mutants, gives rise to quality control-mediated routing of Ena1p to the vacuole.

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