Qualitative and Quantitative Multiplexed Proteomic Analysis of Complex Yeast Protein Fractions That Modulate the Assembly of the Yeast Prion Sup35p

Virginie Redeker,Chris Hughes,Johannes P C Vissers,Ronald Melki,Jimmy Savistchenko,Jie Zheng

doi:10.1371/journal.pone.0023659

Abstract

BackgroundThe aggregation of the baker's yeast prion Sup35p is at the origin of the transmissible [PSI+] trait. We and others have shown that molecular chaperones modulate Sup35p aggregation. However, other protein classes might be involved in [PSI+] formation.ResultsWe designed a functional proteomic study that combines two techniques to identify modulators of Sup35p aggregation and describe the changes associated to [PSI+] formation. The first allows measuring the effect of fractionated Saccharomyces cerevisiae cytosolic extracts from [PSI+] and [psi−] yeast cells on Sup35p assembly. The second is a multiplex qualitative and quantitative comparison of protein composition of active and inactive fractions using a gel-free and label-free LC-MS approach. We identify changes in proteins involved in translation, folding, degradation, oxido-reduction and metabolic processes.ConclusionOur functional proteomic study provides the first inventory list of over 300 proteins that directly or indirectly affect Sup35p aggregation and [PSI+] formation. Our results highlight the complexity of the cellular changes accompanying [PSI+] formation and pave the way for in vitro studies aimed to document the effect of individual and/or combinations of proteins identified here, susceptible of affecting Sup35p assembly.

Highlights

Infectious proteins aggregation is at the origin of neurodegeneration in higher vertebrates [1]
Yeast prions constitute good models to document the mechanism of protein aggregation and conformational conversion propagation observed in conformational neurological diseases
The cytosolic protein fractionation coupled to the functional studies and qualitative and quantitative multiplexed label-free proteomic analysis we present here was intended to identify proteins that modulate Sup35p assembly and [PSI+] emergence and propagation in yeast cells

Summary

Introduction

Infectious proteins (prions) aggregation is at the origin of neurodegeneration in higher vertebrates [1]. Prions are at the origin of dominant phenotypic traits that are inherited in a non-Mendelian manner and are transmissible by cytoduction in the yeast Saccharomyces cerevisiae [4]. Three such traits are actively studied: [PSI+], [URE3] and [PIN+]. Yeast prions N-terminal moieties that are essential for prion aggregation and propagation are unusually rich in glutamine and asparagine residues. They resemble in that to huntingtin which aggregation is involved in the neurodegenerative Huntington’s disease. Other protein classes might be involved in [PSI+] formation

Methods

Results

Conclusion