Primate Chaperones Hsc70 (Constitutive) and Hsp70 (Induced) Differ Functionally in Supporting Growth and Prion Propagation in Saccharomyces cerevisiae

Abstract

Hsp70's are highly conserved essential protein chaperones that assist protein folding and prevent protein aggregation. They have modular structures consisting of ATPase, substrate-binding, and C-terminal domains. Substrate binding and release is regulated by ATP hydrolysis and nucleotide exchange, which in turn are regulated by cochaperones. Eukaryotes have constitutive (Hsc70) and stress-inducible (iHsp70) isoforms, but their functions have not been systematically compared. Using a yeast system to evaluate heterologous Hsp70's we find that primate Hsc70 supported growth but iHsp70 did not. Plant Hsc70 and iHsp70 counterparts behaved similarly, implying evolutionary conservation of this distinction. Swapping yeast and primate Hsp70 domains showed that (i) the Hsc70-iHsp70 distinction resided in the ATPase domain, (ii) substrate-binding domains of Hsp70's within and across species functioned similarly regarding growth, (iii) C-terminal domain function was important for growth, and (iv) Hsp70 functions important for cell growth and prion propagation were separable. Enzymatic analysis uncovered a correlation between substrate affinity and prion phenotype and showed that ATPase and protein-folding activities were generally similar. Our data support a view that intrinsic activities of Hsp70 isoforms are comparable, and functional differences in vivo lie mainly in complex interactions of Hsp70 with cochaperones.

Functional analysis of Hsp70 superfamily proteins of rice (Oryza sativa)

“…Functional distinction could be ascribed to the interaction of Hsp70 with its co-chaperones. Tutar et al (2006) also showed that growth of a yeast mutant in cytosolic Hsp70s was faster when supported by its own Ssa1p than Arabidopsis or primate Hsc70. Partial complementation signifies that though Hsp70 are evolutionarily highly conserved, species specificity still plays a role.…”

Single methyl group determines prion propagation and protein degradation activities of yeast heat shock protein (Hsp)-70 chaperones Ssa1p and Ssa2p

“…The NBD, however, which regulates substrate binding by the SBD, is much more important for determining specific activities of different Hsp70s in vivo. For example, a majority of Hsp70 alterations that affect yeast prion propagation are in the NBD, and the capacity of Hsp70 to support yeast viability is governed primarily by the NBD (4,(29)(30)(31). These observations suggest that the regulation of Hsp70 activity, and not substrate binding per se, is the critical parameter for determining specificity of function.…”

“…Humans and Saccharomyces cerevisiae each have six canonical cytosolic Hsp70s. Their functions overlap to the extent that human Hsp70 (Hsc70) can support viability of yeast lacking its essential Ssa Hsp70 subfamily (4). Similarly, exogenously expressed heterologous Hsp70 protects animals from various stresses (5,6).…”

“…Similarly, exogenously expressed heterologous Hsp70 protects animals from various stresses (5,6). Nevertheless, even highly homologous isoforms display clearly distinct functions (4,(7)(8)(9)(10)(11). Whereas the amplification of Hsp70 genes has long been considered advantageous for allowing regulation of spatiotemporal abundance of Hsp70 according to cellular need for its chaperone function, the ability of Hsp70 to function distinctly implies that expansion of Hsp70 genes also provides an advantage as a source of functional diversity.…”

Functionally Redundant Isoforms of a Yeast Hsp70 Chaperone Subfamily Have Different Antiprion Effects

“…Nevertheless, it has begun to be appreciated that different isoforms have specialized roles or possess different functions (Tutar et al 2006;Daugaard et al 2007;Genevaux et al 2007;Brocchieri et al 2008). Here we show that each of the four yeast cytosolic Ssa Hsp70's function differently with regard to propagation of yeast prions.…”