Primary Sequence That Determines the Functional Overlap between Mitochondrial Heat Shock Protein 70 Ssc1 and Ssc3 of Saccharomyces cerevisiae

Abstract

The evolutionary diversity of the HSP70 gene family at the genetic level has generated complex structural variations leading to altered functional specificity and mode of regulation in different cellular compartments. By utilizing Saccharomyces cerevisiae as a model system for better understanding the global functional cooperativity between Hsp70 paralogs, we have dissected the differences in functional properties at the biochemical level between mitochondrial heat shock protein 70 (mtHsp70) Ssc1 and an uncharacterized Ssc3 paralog. Based on the evolutionary origin of Ssc3 and a high degree of sequence homology with Ssc1, it has been proposed that both have a close functional overlap in the mitochondrial matrix. Surprisingly, our results demonstrate that there is no functional cross-talk between Ssc1 and Ssc3 paralogs. The lack of in vivo functional overlap is due to altered conformation and significant lower stability associated with Ssc3. The substrate-binding domain of Ssc3 showed poor affinity toward mitochondrial client proteins and Tim44 due to the open conformation in ADP-bound state. In addition to that, the nucleotide-binding domain of Ssc3 showed an altered regulation by the Mge1 co-chaperone due to a high degree of conformational plasticity, which strongly promotes aggregation. Besides, Ssc3 possesses a dysfunctional inter-domain interface thus rendering it unable to perform functions similar to generic Hsp70s. Moreover, we have identified the critical amino acid sequence of Ssc1 and Ssc3 that can “make or break” mtHsp70 chaperone function. Together, our analysis provides the first evidence to show that the nucleotide-binding domain of mtHsp70s plays a critical role in determining the functional specificity among paralogs and orthologs across kingdoms.

Biology of the Heat Shock Response and Protein Chaperones: Budding Yeast (Saccharomyces cerevisiae) as a Model System

“…However, the disruption of SSC3 in the conditional ssc1-3 strain resulted in heightened cold sensitivity when grown on glycerol compared to the ssc1-3 strain alone, suggesting a role for Ssc3 in protection against cold stress when Ssc1 is partially defective (16). Contrary to the above-described evidence, recent data showed that SSC3, when overexpressed in the ssc1⌬ strain complemented with the SSC1 gene on a URA3-marked plasmid, cannot support growth after counterselection for the loss of the complementing plasmid on 5-fluoroorotic acid (5-FOA) (330). These results suggest that SSC3 may not be competent to replace all the biological functions of SSC1 when the latter is absent, as opposed to being compromised by a point mutation.…”

“…In support of this conjecture, fluorescence anisotropy measurements showed that Ssc3 exhibited a lower affinity with a generic Hsp70-binding peptide than Ssc1. Using chimeras of Ssc1 and Ssc3, the D-helix within the SBD of both proteins was shown to be responsible for their differential affinities for client proteins (330). These functional differences may explain the observation that Ssc3 is present in only a small subset of closely related fungi, including S. cerevisiae, Saccharomyces bayanus, and Candida glabrata.…”

Unraveling the Intricate Organization of Mammalian Mitochondrial Presequence Translocases: Existence of Multiple Translocases for Maintenance of Mitochondrial Function

“…The initial translocation across the machinery is inner membrane potential dependent, and the final step is driven by ATPase activity of the import motor (11)(12)(13). The mitochondrial Hsp70 (mtHsp70), with the aid of accessory factors such as J-proteins, plays a critical central role during the process.…”

Molecular Insights Revealing Interaction of Tim23 and Channel Subunits of Presequence Translocase

“…For preparation of antibody to Tim21, a fragment corresponding to amino acids 103 to 229 was cloned into the pET-3a vector and purified from the supernatant fraction using Escherichia coli. BL21(DE3) cells, as previously described (51). For preparation of antibody to Pam17, the ORF was cloned in the pET-3a vector and protein was reconstituted from the pellet fraction using the C41 expression strain according to previously published protocols (51).…”

“…BL21(DE3) cells, as previously described (51). For preparation of antibody to Pam17, the ORF was cloned in the pET-3a vector and protein was reconstituted from the pellet fraction using the C41 expression strain according to previously published protocols (51). To raise antibodies against Tim17, a C-terminal peptide (CEAPSSQPLQA) was used for immunization.…”