Efficient ER exit and vacuole targeting of yeast Sna2p require two tyrosine-based sorting motifs : the proteome of lipid bodies

(eng) SNA proteins (Sensitive to Na+) 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 1 (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 (LBs), 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 demonstrated to be functional at two different steps of the secretory pathway, ER exit and Golgi-to-vacuole transport. Furthermore, the LB localization of Sna2p mutants led us to study further those subcellular structures. Different proteomic studies on LBs from yeast have been published previously. However, all of them were using a SDS-PAGE approach : LB proteins were separated on a gel and analyzed by mass spectrometry. Here, we used a gel-free approach. Proteins from purified LBs were digested by trypsin and the peptides separated by reverse phase liquid chromatography before to be submitted to mass spectrometry analysis. This technique allowed us to find all the known LB proteins identified in previous mass spectromotry studies, but also new candidates.