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1.  Identification of protein complexes with quantitative proteomics in S. cerevisiae 
Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach.
doi:10.3791/1225
PMCID: PMC2789102  PMID: 19262458
2.  Lipids as conductors in the orchestra of life 
The lipid phosphatidic acid is an important metabolic intermediate in the biosynthesis of lipids in all eukaryotic cells, but it is even more than that. Phosphatidic acid is emerging as a lipid that is both composer and conductor, where in addition to its role as biosynthetic precursor (composer) it is also a potent signaling molecule (conductor) that integrates membrane biogenesis with nutrient sensing and cell growth. This article discusses recent advances in yeast that give praise for phosphatidic acid as one of life's conductors.
doi:10.3410/B4-4
PMCID: PMC3270589  PMID: 22312416
3.  Inheritance of cortical ER in yeast is required for normal septin organization 
The Journal of Cell Biology  2007;179(3):467-483.
How cells monitor the distribution of organelles is largely unknown. In budding yeast, the largest subdomain of the endoplasmic reticulum (ER) is a network of cortical ER (cER) that adheres to the plasma membrane. Delivery of cER from mother cells to buds, which is termed cER inheritance, occurs as an orderly process early in budding. We find that cER inheritance is defective in cells lacking Scs2, a yeast homologue of the integral ER membrane protein VAP (vesicle-associated membrane protein–associated protein) conserved in all eukaryotes. Scs2 and human VAP both target yeast bud tips, suggesting a conserved action of VAP in attaching ER to sites of polarized growth. In addition, the loss of either Scs2 or Ice2 (another protein involved in cER inheritance) perturbs septin assembly at the bud neck. This perturbation leads to a delay in the transition through G2, activating the Saccharomyces wee1 kinase (Swe1) and the morphogenesis checkpoint. Thus, we identify a mechanism involved in sensing the distribution of ER.
doi:10.1083/jcb.200708205
PMCID: PMC2064793  PMID: 17984322
4.  The Role of Subunit Assembly in Peripherin-2 Targeting to Rod Photoreceptor Disk Membranes and Retinitis Pigmentosa 
Molecular Biology of the Cell  2003;14(8):3400-3413.
Peripherin-2 is a member of the tetraspanin family of membrane proteins that plays a critical role in photoreceptor outer segment disk morphogenesis. Mutations in peripherin-2 are responsible for various retinal degenerative diseases including autosomal dominant retinitis pigmentosa (ADRP). To identify determinants required for peripherin-2 targeting to disk membranes and elucidate mechanisms underlying ADRP, we have generated transgenic Xenopus tadpoles expressing wild-type and ADRP-linked peripherin-2 mutants as green fluorescent fusion proteins in rod photoreceptors. Wild-type peripherin-2 and P216L and C150S mutants, which assemble as tetramers, targeted to disk membranes as visualized by confocal and electron microscopy. In contrast the C214S and L185P mutants, which form homodimers, but not tetramers, were retained in the rod inner segment. Only the P216L disease mutant induced photoreceptor degeneration. These results indicate that tetramerization is required for peripherin-2 targeting and incorporation into disk membranes. Tetramerization-defective mutants cause ADRP through a deficiency in wild-type peripherin-2, whereas tetramerization-competent P216L peripherin-2 causes ADRP through a dominant negative effect, possibly arising from the introduction of a new oligosaccharide chain that destabilizes disks. Our results further indicate that a checkpoint between the photoreceptor inner and outer segments allows only correctly assembled peripherin-2 tetramers to be incorporated into nascent disk membranes.
doi:10.1091/mbc.E03-02-0077
PMCID: PMC181576  PMID: 12925772

Results 1-4 (4)