Proteomics of Saccharomyces cerevisiae Organelles

Abstract

Knowledge of the subcellular localization of proteins is indispensable to understand their physiological roles. In the past decade, 18 studies have been performed to analyze the protein content of isolated organelles from Saccharomyces cerevisiae. Here, we integrate the data sets and compare them with other large scale studies on protein localization and abundance. We evaluate the completeness and reliability of the organelle proteomics studies. Reliability depends on the purity of the organelle preparations, which unavoidably contain (small) amounts of contaminants from different locations. Quantitative proteomics methods can be used to distinguish between true organellar constituents and contaminants. Completeness is compromised when loosely or dynamically associated proteins are lost during organelle preparation and also depends on the sensitivity of the analytical methods for protein detection. There is a clear trend in the data from the 18 organelle proteomics studies showing that proteins of low abundance frequently escape detection. Proteins with unknown function or cellular abundance are also infrequently detected, indicating that these proteins may not be expressed under the conditions used. We discuss that the yeast organelle proteomics studies provide powerful lead data for further detailed studies and that methodological advances in organelle preparation and in protein detection may help to improve the completeness and reliability of the data. Molecular & Cellular Proteomics 9:431-445, 2010.

The plasma membrane proteome of Medicago truncatula roots as modified by arbuscular mycorrhizal symbiosis

“…Consequently, both the enrichment in plasmalemmic proteins and depletion of contaminants also needs to be quantified by comparing protein abundances in crude and purified fractions (Yadeta et al 2013). The rationale behind this approach is that proteins physically associated with an organelle become enriched upon purification, while contaminants are depleted (Wiederhold et al 2010). Digital immunoblotting was then carried out on the crude microsomal fractions of M. truncatula roots using the protein list published by Abdallah et al (2014).…”

Ordered Assembly of Heat Shock Proteins, Hsp26, Hsp70, Hsp90, and Hsp104, on Expanded Polyglutamine Fragments Revealed by Chemical Probes

“…Data-dependent MS/MS spectra on the 5 most intense ions from each full MS scan were collected (relative CE ϳ35%). All mass spectra acquired were analyzed and matched to the S. cerevisiae database (6817 entries, 2008 release) appended with decoy reverse sequences and human polyQ protein using X!Tandem/Trans-Proteomic Pipeline (TPP) software suite (38). All peptides and proteins with a PeptideProphet and ProteinProphet probability score of Ͼ0.9 (fdr Ͻ2%) were considered positive identifications.…”

“…Immunoprecipitations and Western Blots-The protocol for the collection of yeast fractions suitable for proteomics was described by Wiederhold et al (38). Briefly, yeast cells of a 30 ml exponentially growing culture were harvested by centrifugation at 3000 revolutions/min for 5 min at 18°C, suspended in lysis buffer (Tris-HCl 50 mM, 200 mM sorbitol, 1 mM MgCl 2 , 0.1% Tween 20, 1 mM PMSF, and protease mixture inhibitor) and disrupted by vortexing with glass beads (6 ϫ 30 s, 1 min ice in between).…”

The proteomics of lipid droplets: structure, dynamics, and functions of the organelle conserved from bacteria to humans

“…The recent prevalence of lipid metabolic diseases has motivated research on lipid metabolism in yeast, especially the study of lipid droplets (also referred to as lipid particles and lipid bodies). During the last decade, proteomic studies of yeast lipid droplets have shed new light on the role of lipid droplets in lipid synthesis ( 34 ) and membrane traffi cking ( 35,36,87 ), and they have identifi ed some proteins relevant to lipid metabolic disease in humans, such as the homolog of seipin ( 35 ). Mutants of seipin are related to lipodystrophy in humans ( 88 ) and to supersized lipid droplets in yeast ( 89 ).…”