Identification of protein complexes with quantitative proteomics in S. cerevisiae

Summary

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

Abstract

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.

Protocol

Materials and Methods

1. Yeast strains
   • All strains used in this study are based on the BY4742 background. The SILAC control strain (Mat a, his3, leu2, ura3, lys2 and arg4::G418) was made by mating arg4 deletion strain (Mat a, his3, ura3, leu2, and arg4::G418) to BY4742 (Mat alpha, his3, leu2, ura3 and lys2), and the meiotic haploids were obtained by tetrad dissection. Therefore, the control strain is an auxotroph for lysine and arginine. The bait strain was made by PCR-mediated homologous recombination using the vector pBS1479. Therefore, the epitope tag used in this study is the tandem affinity purifica....

Discussion

The aliquots saved during the purification procedure should include (1) pre-cleared cell lysate, (2) bound fraction, (3) unbound fraction, and (4) eluted fraction. We recommend analyzing the protein contents of the above aliquots by Western blotting using anti-TAP antibody or silver staining to reflect the binding and eluting efficiency of the experiment. Examples of a silver stained gel and a blot are shown in Figure 1.

Since SILAC provides us with unbiased and quantified measurements of pro.......