The Role of Djp1 in Import of the Mitochondrial Protein Mim1 Demonstrates Specificity between a Cochaperone and Its Substrate Protein

Abstract

A special group of mitochondrial outer membrane proteins spans the membrane once, exposing soluble domains to both sides of the membrane. These proteins are synthesized in the cytosol and then inserted into the membrane by an unknown mechanism. To identify proteins that are involved in the biogenesis of the single-span model protein Mim1, we performed a high-throughput screen in yeast. Two interesting candidates were the cytosolic cochaperone Djp1 and the mitochondrial import receptor Tom70. Our results indeed demonstrate a direct interaction of newly synthesized Mim1 molecules with Tom70. We further observed lower steady-state levels of Mim1 in mitochondria from djp1⌬ and tom70 tom71⌬ cells and massive mislocalization of overexpressed GFP-Mim1 to the endoplasmic reticulum in the absence of Djp1. Importantly, these phenotypes were observed specifically for the deletion of DJP1 and were not detected in mutant cells lacking any of the other cytosolic cochaperones of the Hsp40 family. Furthermore, the djp1⌬ tom70⌬ tom71⌬ triple deletion resulted in a severe synthetic sick/lethal growth phenotype. Taking our results together, we identified Tom70 and Djp1 as crucial players in the biogenesis of Mim1. Moreover, the involvement of Djp1 provides a unique case of specificity between a cochaperone and its substrate protein.

Genome-Wide Screens in Saccharomyces cerevisiae Highlight a Role for Cardiolipin in Biogenesis of Mitochondrial Outer Membrane Multispan Proteins

“…Interestingly, our visual screen could not identify mutants that result in mistargeting of Om14 to the endoplasmic reticulum (ER). This is in contrast to previous studies with MOM single-span proteins such as Mim1 or Gem1 where deletions of DJP1 (34) or SPF1 (33), respectively, resulted in mislocalization of the mitochondrial protein to the ER. Hence, it seems that, whereas the single-span proteins can, in principle, under certain conditions, also get integrated into the ER membrane, the multispan proteins lack this capacity or else might get efficiently and rapidly degraded in the event that they do.…”

“…In the latter cases, mitochondria were usually aggregated, probably due to the elevated levels of GFP-Om14 in the MOM. However, in contrast to mutants that affect targeting of MOM single-span proteins (33,34), none of the inspected strains displayed mistargeting to other organelles. Manually annotating the resulting images for changes in localization or automatically extracting intensity data, we could find 146 strain backgrounds with altered intensity and/or altered localization of the GFP construct (see Table S1 in the supplemental material).…”

“…3A). We have previously used a similar method to successfully monitor the membrane insertion of the MOM single-span protein Mim1 (34). When the Om14 fusion protein is integrated into the MOM with its native topology (31), Ura3 is exposed to the cytosol (where it is functional), whereas SL17 is hidden in the IMS and is not exposed to the ubiquitinproteasome system (Fig.…”

Partial dispensability of Djp1's J domain in peroxisomal protein import in Saccharomyces cerevisiae results from genetic redundancy with another class II J protein, Caj1

“…Peroxisomal targeting signal 1 (PTS1), with the sequence Ser/Ala/Cys, Lys/Arg/His, Leu/Met/Ile is found on most proteins destined to be imported into the peroxisomal matrix (Smith and Aitchison 2013). Additionally, Djp1 is specifically required for biogenesis of outer mitochondrial proteins Mim1 and Mim2 in S. cerevisiae (Papic et al 2013).…”

Dynamic organization of the mitochondrial protein import machinery

“…Mitochondrial precursor proteins can be bound to cytosolic chaperones and possibly further factors. The specific interaction of chaperones or putative targeting factors with the membrane-bound protein import machinery of mitochondria will be an attractive area of research (Young et al, 2003;Schmidt et al, 2011;Papić et al, 2013). The majority of mitochondrial proteins can be imported in a post-translational manner, i.e.…”