Inactivation of the Mitochondrial Heat Shock Protein Zim17 Leads to Aggregation of Matrix Hsp70s Followed by Pleiotropic Effects on Morphology and Protein Biogenesis

Previous efforts aimed at the biochemical characterization of chloroplast HSP70B were hampered by the observation that recombinant HSP70B was inactive, i.e. incompetent of interacting with its nucleotide exchange factor CGE1. In addition, because heterologously expressed mitochondrial Hsp70 was inactive unless coexpressed with the escort protein Hep1, we wondered whether homologs of Hep1 existed in the chloroplast. Data base searches revealed that algae and higher plants indeed encode at least two HEP homologs, one predicted to be targeted to mitochondria, the others to chloroplasts. Using Chlamydomonas reinhardtii as plant model organism we demonstrate that this alga encodes an HEP homolog (termed HEP2) that is localized to the stroma. HEP2 is expressed constitutively as a low abundance protein with an apparent molecular mass of ϳ21 kDa. In cell extracts HEP2 interacts with HSP70B in an ATP-dependent fashion. Coexpression of HSP70B with HEP2 in Escherichia coli yielded high levels of CGE1-binding competent HSP70B, which also displayed ATPase activity. Inactive HSP70B was more prone to proteolysis than active HSP70B. Although inactive HSP70B interacted with HEP2, it could not be activated. Active HSP70B remained active for 48 h in the absence of HEP2, suggesting that HEP2 was not involved in maintaining HSP70B in an active state. However, some HSP70B expressed as a fusion protein with an N-terminal extension was activated when HEP2 was present during cleavage of the fusion protein, suggesting that in vivo HEP2 might be required for de novo folding of HSP70B after transit peptide cleavage.

Section: Algae and Higher Plants Encode Hep Homologs Predicted Tomentioning

confidence: 99%

Assistance for a Chaperone

Willmund

Hinnenberger

Nick

et al. 2008

“…Recent data identified further interactors of the chaperone. Zinc finger motif protein of 17 kDa (Zim17, also termed mtHsp70 escort protein 1 (Hep1)) supports the folding and function of the chaperone (32)(33)(34)(35)(36)(37). Furthermore, mtHsp70 interacts with Mss51 and Cox4 to promote the biogenesis of the cytochrome c oxidase (complex IV of the respiratory chain) (38,39).…”

mentioning

confidence: 99%

Mitochondrial Heat Shock Protein (Hsp) 70 and Hsp10 Cooperate in the Formation of Hsp60 Complexes

Böttinger

Oeljeklaus

Guiard

et al. 2015

Self Cite

“…However, a direct analysis of Zim17 function under these conditions was difficult due to the accumulation of severe secondary defects caused by the aggregation of mtHsp70s. Our analysis of a previously published conditional (temperature-sensitive) mutant strain, zim17-2 (26), exhibited that the mutant protein was virtually undetectable in isolated mitochondria (data not shown). This behavior is most likely due to the presence of an additional mutation in its presequence that interfered with its mitochondrial import, whereas the overall stability of the mutant protein did not seem to be affected (data not shown).…”

Section: Discussionmentioning

confidence: 66%

“…The mutant zim17-3a gene sequence was amplified from the plasmid pFLzim17-Ts3-CEN obtained from the yeast strain BGYFomp3-C1 (26). The novel mutant zim17-3b was generated by site-directed mutagenesis using PCR for the replacement of the serine residue at position 79 of mutant zim17-3a with an asparagine.…”

Section: Methodsmentioning

confidence: 99%

“…Indeed, a complete loss of Zim17 in yeast deletion mutants (zim17⌬) resulted in the aggregation of the mitochondrial chaperones Ssc1 and Ssq1. Concomitantly, zim17⌬ cells showed severe phenotypic defects like the inability to grow at elevated temperatures and a high tendency to generate the respiratory deficient petite phenotype (22,23,25,26). This phenotype has been explained by the long term accumulation of secondary defects that are related to a loss of chaperone activity.…”

Section: The Yeast Protein Zim17 Belongs To a Unique Class Of Co-chapmentioning

confidence: 99%

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The Functional Interaction of Mitochondrial Hsp70s with the Escort Protein Zim17 Is Critical for Fe/S Biogenesis and Substrate Interaction at the Inner Membrane Preprotein Translocase

Lewrenz

Rietzschel²,

Guiard

et al. 2013

Self Cite

Background: Zim17 represents a Hsp70-interacting protein residing in the mitochondrial matrix compartment. Results: Conditional zim17 mutants show functional defects in essential mtHsp70 activities, biosynthesis of Fe/S clusters, and interaction with newly imported substrate proteins. Conclusion: Zim17 exerts a direct regulative function on Hsp70 independent of its aggregation-protective role. Significance: Identification of novel co-chaperone mechanism unravels complex functional regulation of Hsp70 chaperones.