Multiple Hsp70 Isoforms in the Eukaryotic Cytosol: Mere Redundancy or Functional Specificity?

Hsp70 molecular chaperones play a variety of functions in every organism, cell type and organelle, and their activities have been implicated in a number of human pathologies, ranging from cancer to neurodegenerative diseases. The functions, regulations and structure of Hsp70s were intensively studied for about three decades, yet much still remains to be learned about these essential folding enzymes. Genome sequencing efforts revealed that most genomes contain multiple members of the Hsp70 family, some of which co-exist in the same cellular compartment. For example, the human cytosol and nucleus contain six highly homologous Hsp70 proteins while the yeast Saccharomyces cerevisiae contains four canonical Hsp70s and three fungal-specific ribosome-associated and specialized Hsp70s. The reasons and significance of the requirement for multiple Hsp70s is still a subject of debate. It has been postulated for a long time that these Hsp70 isoforms are functionally redundant and differ only by their spatio-temporal expression patterns. However, several studies in yeast and higher eukaryotic organisms challenged this widely accepted idea by demonstrating functional specificity among Hsp70 isoforms. Another element of complexity is brought about by specific cofactors, such as Hsp40s or nucleotide exchange factors that modulate the activity of Hsp70s and their binding to client proteins. Hence, a dynamic network of chaperone/co-chaperone interactions has evolved in each organism to efficiently take advantage of the multiple cellular roles Hsp70s can play. We summarize here our current knowledge of the functions and regulations of these molecular chaperones, and shed light on the known functional specificities among isoforms

Function of SSA Subfamily of Hsp70 Within and Across Species Varies Widely in Complementing Saccharomyces cerevisiae Cell Growth and Prion Propagation

BACKGROUND:The cytosol of most eukaryotic cells contains multiple highly conserved Hsp70 orthologs that differ mainly by their spatio-temporal expression patterns. Hsp70s play essential roles in protein folding, transport or degradation, and are major players of cellular quality control processes. However, while several reports suggest that specialized functions of Hsp70 orthologs were selected through evolution, few studies addressed systematically this issue. METHODOLOGY/PRINCIPAL FINDINGS:We compared the ability of Ssa1p-Ssa4p from Saccharomyces cerevisiae and Ssa5p-Ssa8p from the evolutionary distant yeast Yarrowia lipolytica to perform Hsp70-dependent tasks when expressed as the sole Hsp70 for S. cerevisiae in vivo. We show that Hsp70 isoforms (i) supported yeast viability yet with markedly different growth rates, (ii) influenced the propagation and stability of the [PSI(+)] and [URE3] prions, but iii) did not significantly affect the proteasomal degradation rate of CFTR. Additionally, we show that individual Hsp70 orthologs did not induce the formation of different prion strains, but rather influenced the aggregation properties of Sup35 in vivo. Finally, we show that [URE3] curing by the overexpression of Ydj1p is Hsp70-isoform dependent. CONCLUSION/SIGNIFICANCE:Despite very high homology and overlapping functions, the different Hsp70 orthologs have evolved to possess distinct activities that are required to cope with different types of substrates or stress situations. Yeast prions provide a very sensitive model to uncover this functional specialization and to explore the intricate network of chaperone/co-chaperone/substrates interactions

Hiding in plain sight: vesicle-mediated export and transmission of prion-like proteins

International audienceInfectious proteins or prions are non-native confor-mations of proteins that are the causative agents of devastating neurodegenerative diseases in humans and heritable traits in filamentous fungi and yeasts. Prion proteins form highly ordered self-perpetuating fibrillar aggregates that traffic vertically and horizontally from cell to cell. The spreading of these infectious entities relies on different mechanisms, among which the extracellular vesicles (EV)-mediated traffic. The prion form of the yeast Saccharomyces cerevisiae Sup35p translation terminator causes the [PSI + ] nonsense suppression phenotype. This fascinating biological model helped us shape our understanding of the mechanisms of formation, propagation and elimination of infectious protein aggregates. We discovered that Sup35p is exported via EV, both in its soluble and aggregated infectious states. We recently reported that high amounts of Sup35p prion particles are exported to the yeast periplasm via periplasmic vesicles (PV) in glucose-starved cells. EV and PV are different in terms of size and protein content, and their export is inversely regulated by glucose availability in the growth medium. We believe these are important observations that should make us revise our current view on the way yeast prions propagate. Hence, I propose several hypotheses as to the significance of these observations for the transmission of yeast prions. I also discuss how yeast could be used as a powerful tracta-ble biological model to investigate the molecular mechanisms of vesicle-mediated export of pathological protein aggregates implicated in neurodegenerative diseases

Extracellular vesicles and the propagation of yeast prions

International audienc

Extracellular Vesicles-Encapsulated Yeast Prions and What They Can Tell Us about the Physical Nature of Propagons

International audienceThe yeast Saccharomyces cerevisiae hosts an ensemble of protein-based heritable traits, most of which result from the conversion of structurally and functionally diverse cytoplasmic proteins into prion forms. Among these, [PSI + ], [URE3] and [PIN + ] are the most well-documented prions and arise from the assembly of Sup35p, Ure2p and Rnq1p, respectively, into insoluble fibrillar assemblies. Yeast prions propagate by molecular chaperone-mediated fragmentation of these aggregates, which generates small self-templating seeds, or propagons. The exact molecular nature of propagons and how they are faithfully transmitted from mother to daughter cells despite spatial protein quality control are not fully understood. In [PSI + ] cells, Sup35p forms detergent-resistant assemblies detectable on agarose gels under semi-denaturant conditions and cytosolic fluorescent puncta when the protein is fused to green fluorescent protein (GFP); yet, these macroscopic manifestations of [PSI + ] do not fully correlate with the infectivity measured during growth by the mean of protein infection assays. We also discovered that significant amounts of infectious Sup35p particles are exported via extracellular (EV) and periplasmic (PV) vesicles in a growth phase and glucose-dependent manner. In the present review, I discuss how these vesicles may be a source of actual propagons and a suitable vehicle for their transmission to the bud

The Yarrowia lipolytica orthologs of Sup35p assemble into thioflavin T-negative amyloid fibrils

International audienceThe translation terminator Sup35p assembles into self-replicating fibrillar aggregates that are responsible for the [PSI þ ] prion state. The Q/N-rich N-terminal domain together with the highly charged middle-domain (NM domain) drive the assembly of Sup35p into amyloid fibrils in vitro. NM domains are highly divergent among yeasts. The ability to convert to a prion form is however conserved among Sup35 orthologs. In particular, the Yarrowia lipolytica Sup35p stands out with an exceptionally high prion conversion rate. In the present work, we show that different Yarrowia lipolytica strains contain one of two Sup35p orthologs that differ by the number of repeats within their NM domain. The Y. lipolytica Sup35 proteins are able to assemble into amyloid fibrils. Contrary to S. cerevisiae Sup35p, fibrils made of full-length or NM domains of Y. lipolytica Sup35 proteins did not bind Thioflavin-T, a well-known marker of amyloid aggregates

Spécificités fonctionnelles des Hsp70 cytoplasmiques chez la levure

Les Hsp70 constituent une famille de chaperons moléculaires ubiquitaires qui jouent des rôles essentiels dans le repliement, le transport ou la dégradation des protéines. Le cytoplasme des cellules eucaryotes contient plusieurs paralogues de Hsp70 fortement conservés qui diffèrent essentiellement par leur expression spatio-temporelle. Plusieurs travaux suggèrent que ces paralogues ont des spécificités fonctionnelles que nous avons cherché à mettre en lumière et caractériser par des approches génétiques. Dans une première étude, nous avons comparé les activités des Hsp70 des levures Saccharomyces cerevisiae (Ssa1-4) et Yarrowia lipolytica (Ssa5-8) lorsqu'elles sont exprimées comme unique Hsp70 chez S. cerevisiae. Nous avons montré que ces Hsp70: 1) assurent la viabilité des cellules mais avec des taux de croissance très différents; 2) ont des effets variables sur la propagation et la stabilité des prions [URE3] et [PSI+]; et 3) permettent la dégradation protéasomale de CFTR avec des cinétiques comparables. Dans une seconde étude, nous avons montré que la formation de biofilms chez la levure dépend de la machinerie Hsp70 qui contrôle, via des voies distinctes, l'expression, la maturation et le recyclage d'une adhésine de surface (Flo11) requise pour ce processus. Enfin, nous avons construit et caractérisé des mutants de Y. lipolytica dans lesquels un ou plusieurs gènes codant des chaperons moléculaires ou acteurs de la protéostase (e.g. Hsp70, Hsp104, CHIP) ont été invalidés. Malgré une forte homologie et une redondance fonctionnelle, les Hsp70 possèdent des propriétés distinctes permettant aux cellules de faire face à différents types de substrats et de conditions de stressHsp70 are a highly conserved family of ubiquitous molecular chaperones that play essential roles in protein folding, transport or degradation. The cytosol of most eukaryotic cells contains multiple highly conserved Hsp70 orthologs that differ mainly by their spatio-temporal expression patterns. While several reports suggest that specialized functions of Hsp70 orthologs were selected through evolution, few studies addressed systematically this issue. First, we compared the ability of Ssa1-Ssa4 from Saccharomyces cerevisiae and Ssa5-Ssa8 from the evolutionary distant yeast Yarrowia lipolytica to perform Hsp70-dependent tasks when expressed as the sole Hsp70 for S. cerevisiae in vivo. We showed that Hsp70 isoforms: 1) supported yeast viability yet with markedly different growth rates; 2) influenced the propagation and stability of the [PSI+] and [URE3] prions; but 3) did not significantly affect the proteasomal degradation rate of CFTR. Second, we showed that biofilm formation in yeast depends on the Hsp70 machinery that controls, through distinct pathways, the expression, maturation and recycling of a cell-surface adhesin (Flo11) required for this process. Finally, we constructed and analyzed Y. lipolytica mutants bearing one or multiple deletion(s) in genes encoding molecular chaperones and others proteostasis modulators (e.g. Hsp70, Hsp104, CHIP). Despite very high homology and overlapping functions, the different Hsp70 orthologs have evolved to possess distinct activities that are required to cope with different types of substrates or stress situationsPARIS-AgroParisTech Centre Paris (751052302) / SudocSudocFranceF

TRANSLOCATION ET REPLIEMENT DES PROTEINES DANS LE LUMEN DU RETICULUM ENDOPLASMIQUE CHEZ LA LEVURE

ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF