Interaction of the J-Protein Heterodimer Pam18/Pam16 of the Mitochondrial Import Motor with the Translocon of the Inner Membrane

D’Silva, Patrick; Schilke, Brenda; Hayashi, Masaya; Craig, Elizabeth A.

doi:10.1091/mbc.e07-08-0748

Cited by 70 publications

(103 citation statements)

References 27 publications

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“…However, relatively stable interactions between the J-proteins dimers are well maintained. In contrast, a mild detergent treatment such as digitonin dissolves the intervening membranes without affecting the association of the translocase components (19,20,27,38). The isolated complexes can be resolved through blue native-PAGE and detected by immunoblotting.…”

Section: Formation Of Two Distinct Dimeric Subcomplexes Betweenmentioning

confidence: 99%

Functional Diversity of Human Mitochondrial J-proteins Is Independent of Their Association with the Inner Membrane Presequence Translocase

Sinha

Srivastava

D’Silva

2016

Journal of Biological Chemistry

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Mitochondrial J-proteins play a critical role in governing Hsp70 activity and, hence, are essential for organellar protein translocation and folding. In contrast to yeast, which has a single J-protein Pam18, humans involve two J-proteins, DnaJC15 and DnaJC19, associated with contrasting cellular phenotype, to transport proteins into the mitochondria. Mutation in DnaJC19 results in dilated cardiomyopathy and ataxia syndrome, whereas expression of DnaJC15 regulates the response of cancer cells to chemotherapy. In the present study we have comparatively assessed the biochemical properties of the J-protein paralogs in relation to their association with the import channel. Both DnaJC15 and DnaJC19 formed two distinct subcomplexes with Magmas at the import channel. Knockdown analysis suggested an essential role for Magmas and DnaJC19 in organellar protein translocation and mitochondria biogenesis, whereas DnaJC15 had dispensable supportive function. The J-proteins were found to have equal affinity for Magmas and could stimulate mitochondrial Hsp70 ATPase activity by equivalent levels. Interestingly, we observed that DnaJC15 exhibits bifunctional properties. At the translocation channel, it involves conserved interactions and mechanism to translocate the precursors into mitochondria. In addition to protein transport, DnaJC15 also showed a dual role in yeast where its expression elicited enhanced sensitivity of cells to cisplatin that required the presence of a functional J-domain. The amount of DnaJC15 expressed in the cell was directly proportional to the sensitivity of cells. Our analysis indicates that the differential cellular phenotype displayed by human mitochondrial J-proteins is independent of their activity and association with Magmas at the translocation channel.In eukaryotic cells mitochondria form an essential organelle that plays a central role in cancer, apoptosis, and cytotoxic responses and maintains the overall metabolic homeostasis within the cell (1, 2). More than 98% of the human mitochondrial protein pool is nuclear-encoded and transported into the mitochondria through highly complex import machinery spanning the outer membrane, inner membrane, and the intermembrane space (2, 3). At the inner membrane, TIM23 complex forms the sole gateway for translocation of precursor proteins into the matrix, which accounts for ϳ60% of the total imported proteins (4, 5). The TIM23 complex comprises of two components: membrane-embedded Tim23 core channel and the motor component. Although the initial membrane potential dependent translocation is mediated by the Tim23-Tim17 channel, it is the overall activity of the import motor, which determines the translocation process (3, 4, 6 -10). The import motor contains mtHsp70 as its core, whose function is orchestrated by J-proteins (7,(11)(12)(13). The ADP/ATP states of Hsp70 regulate its interactions with client proteins (14, 15). However, the basal ATPase activity of Hsp70 is not sufficient to drive the reactions efficiently and, thus, requires J-proteins as cofactors ...

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Section: Formation Of Two Distinct Dimeric Subcomplexes Betweenmentioning

confidence: 99%

Functional Diversity of Human Mitochondrial J-proteins Is Independent of Their Association with the Inner Membrane Presequence Translocase

Sinha

Srivastava

D’Silva

2016

Journal of Biological Chemistry

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“…The association of the membrane-bound co-chaperones with the Tim23 channel unit is a prerequisite for the spatially controlled mtHsp70 regulation in mitochondria 27,31,37 . However, beside the translocase-associated portion of the cochaperones Pam16 and Pam18, a significant non-translocase pool of the proteins has been found in mitochondria 11,26,30,31,35 .…”

Section: Discussionmentioning

confidence: 99%

“…However, beside the translocase-associated portion of the cochaperones Pam16 and Pam18, a significant non-translocase pool of the proteins has been found in mitochondria 11,26,30,31,35 . This, situation is now explained by the observed dynamics of the active translocase.…”

Section: Discussionmentioning

confidence: 99%

“…3b, the mutant form of Pam18 displays faster migration in SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analyses and is efficiently recognized by the anti-Pam18 antiserum, lane 1 versus 2). Interestingly, despite the fact that Pam18 and Pam16 are considered to form a stable complex 11,16,26,31 , the inhibitory Pam16 remained translocase-associated in pam18-66 mitochondria. Thus, we concluded that the observed loss of motor function and concomitant instability of the translocation intermediate could be attributed to defective ATPase cycling at the import motor.…”

Section: Import Motor Activity Maintains a Tom/tim23 Intermediatementioning

confidence: 99%

“…4e). It should be noted that although this assay only allows to analyse the integration of subunits, we assume that a release occurs to a similar extent as indicated by the presence of a free pool of Pam16 and Pam18 in mitochondria 11,26,30,31,35 . Thus, our experiments demonstrated that Tim21 and subunits of the PAM complex can be recruited into a precursor-occupied and active translocase, a state that requires import motor activity for formation and maintenance.…”

Section: Import Motor Activity Maintains a Tom/tim23 Intermediatementioning

confidence: 99%

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Remodelling of the active presequence translocase drives motor-dependent mitochondrial protein translocation

Schulz

Rehling

2014

Nat Commun

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Proteins with N-terminal targeting signals are transported across the inner mitochondrial membrane by the presequence translocase. To drive precursor translocation, the Hsp70-import motor associates with the protein-conducting channel of the TIM23 complex. It is unknown how the ATPase cycle of Hsp70 is regulated in the context of a translocating polypeptide chain. Here we establish an assay to monitor protein dynamics in the precursoroccupied presequence translocase and find that regulatory subunits of the import motor, such as the ATPase-stimulating J-protein Pam18, are recruited into the translocation intermediate. The presence of all Hsp70 co-chaperones at the import channel is not sufficient to promote matrix protein import, instead a recharging of the active translocase with Pam18 is required for motor activity. Thus, a replenishment cycle of co-chaperones at the TIM23 complex is an integral part of Hsp70's ATPase cycle at the channel exit site and essential to maintain motordriven mitochondrial protein import.

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Role of membrane contact sites in protein import into mitochondria

et al. 2015

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Mitochondria import more than 1,000 different proteins from the cytosol. The proteins are synthesized as precursors on cytosolic ribosomes and are translocated by protein transport machineries of the mitochondrial membranes. Five main pathways for protein import into mitochondria have been identified. Most pathways use the translocase of the outer mitochondrial membrane (TOM) as the entry gate into mitochondria. Depending on specific signals contained in the precursors, the proteins are subsequently transferred to different intramitochondrial translocases. In this article, we discuss the connection between protein import and mitochondrial membrane architecture. Mitochondria possess two membranes. It is a longstanding question how contact sites between outer and inner membranes are formed and which role the contact sites play in the translocation of precursor proteins. A major translocation contact site is formed between the TOM complex and the presequence translocase of the inner membrane (TIM23 complex), promoting transfer of presequence-carrying preproteins to the mitochondrial inner membrane and matrix. Recent findings led to the identification of contact sites that involve the mitochondrial contact site and cristae organizing system (MICOS) of the inner membrane. MICOS plays a dual role. It is crucial for maintaining the inner membrane cristae architecture and forms contacts sites to the outer membrane that promote translocation of precursor proteins into the intermembrane space and outer membrane of mitochondria. The view is emerging that the mitochondrial protein translocases do not function as independent units, but are embedded in a network of interactions with machineries that control mitochondrial activity and architecture.

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Interaction of the J-Protein Heterodimer Pam18/Pam16 of the Mitochondrial Import Motor with the Translocon of the Inner Membrane

Cited by 70 publications

References 27 publications

Functional Diversity of Human Mitochondrial J-proteins Is Independent of Their Association with the Inner Membrane Presequence Translocase

Functional Diversity of Human Mitochondrial J-proteins Is Independent of Their Association with the Inner Membrane Presequence Translocase

Remodelling of the active presequence translocase drives motor-dependent mitochondrial protein translocation

Role of membrane contact sites in protein import into mitochondria

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