SNA (Sensitive to Na + ) proteins form a membrane protein family, which, in the yeast Saccharomyces cerevisiae, is composed of four members: Sna1p/Pmp3p, Sna2p, Sna3p and Sna4p. In this study, we focused on the 79 residue Sna2p protein. We found that Sna2p is localized in the vacuolar membrane. Directed mutagenesis showed that two functional tyrosine motifs YXXØ are present in the C-terminal region. Each of these is involved in a different Golgi-to-vacuole targeting pathway: the tyrosine 65 motif is involved in adaptor protein (AP-1)-dependent targeting, whereas the tyrosine 75 motif is involved in AP-3-dependent targeting. Moreover, our data suggest that these motifs also play a crucial role in the exit of Sna2p from the endoplasmic reticulum (ER). Directed mutagenesis of these tyrosines led to a partial redirection of Sna2p to lipid bodies, probably because of a decrease in ER exit efficiency. Sna2p is the first yeast protein in which two YXXØ motifs have been identified and both were shown to be functional at two different steps of the secretory pathway, ER exit and Golgi-to-vacuole transport.

Pmp3p-related proteins are highly conserved proteins that exist in bacteria, yeast, nematodes and plants, and its transcript is regulated in response to abiotic stresses, such as low temperature or high salinity. Pmp3p was originally identified in Saccharomyces cerevisiae, and it belongs to the sensitive to Na + (SNA)-protein family, which comprises four members -Pmp3p/Sna1p, Sna2p, Sna3p and Sna4p. Deletion of the PMP3 gene conferred sensitivity to cytotoxic cations, whereas removal of the other SNA genes did not lead to clear phenotypic effects. It has long been believed that Pmp3p-related proteins have a common and important role in the modulation of plasma membrane potential and in the regulation of intracellular ion homeostasis. Here, we show that several growth phenotypes linked to PMP3 deletion can be modulated by the removal of specific genes involved in sphingolipid synthesis. These genetic interactions, together with lipid binding assays and epifluorescence microscopy, as well as other biochemical experiments, suggest that Pmp3p could be part of a phosphoinositide-regulated stress sensor.

Sna4p, a vacuolar membrane protein, belongs to a small family of proteins conserved in plants and fungi. It is transported to the vacuolar membrane via the alkaline phosphatase (ALP) pathway, which bypasses the multivesicular bodies (MVBs). Here, we show that transfer of Sna4p by the ALP route involves the AP-3 adaptor protein complex, which binds to an acidic dileucine sorting signal in the cytoplasmic region of Sna4p. In addition, Sna4p can use the MVB pathway by using a PPPY motif, which is involved in the interaction with ubiquitin ligase Rsp5p. Deletion or mutation of the Sna4p PPPY motif or a low level of Rsp5p inhibits the entrance of Sna4p into MVBs. Sna4p is polyubiquitylated on its only lysine, and Sna4p lacking this lysine shows defective MVB sorting. These data indicate that Sna4p has two functional motifs, one for interaction with the AP-3 complex, followed by entry into the ALP pathway, and one for binding Rsp5p, which directs the protein to the MVB pathway. The presence of these two motifs allows Sna4p to localize to both the vacuolar membrane and the lumen.