Deletion of Mgr2p Affects the Gating Behavior of the TIM23 Complex.

The TIM23 complex is a hub for translocation of preproteins into or across the mitochondrial inner membrane. This dual sorting mechanism is currently being investigated, and in yeast appears to be regulated by a recently discovered subunit, the Mgr2 protein. Deletion of Mgr2p has been found to delay protein translocation into the matrix and accumulation in the inner membrane. This result and other findings suggested that Mgr2p controls the lateral release of inner membrane proteins harboring a stop-transfer signal that follows an N-terminal amino acid signal. However, the mechanism of lateral release is unknown. Here, we used patch clamp electrophysiology to investigate the role of Mgr2p on the channel activity of TIM23. Deletion of Mgr2p decreased normal channel frequency and increased occurrence of a residual TIM23 activity. The residual channel lacked gating transitions but remained sensitive to synthetic import signal peptides. Similarly, a G145L mutation in Tim23p displaced Mgr2p from the import complex leading to gating impairment. These results suggest that Mgr2p regulates the gating behavior of the TIM23 channel.

Role of Tim17 in coupling the import motor to the translocation channel of the mitochondrial presequence translocase.

The majority of mitochondrial proteins use N-terminal presequences for targeting to mitochondria and are translocated by the presequence translocase. During translocation, proteins, threaded through the channel in the inner membrane, are handed over to the import motor at the matrix face. Tim17 is an essential, membrane-embedded subunit of the translocase; however, its function is only poorly understood. Here, we functionally dissected its four predicted transmembrane (TM) segments. Mutations in TM1 and TM2 impaired the interaction of Tim17 with Tim23, component of the translocation channel, whereas mutations in TM3 compromised binding of the import motor. We identified residues in the matrix-facing region of Tim17 involved in binding of the import motor. Our results reveal functionally distinct roles of different regions of Tim17 and suggest how they may be involved in handing over the proteins, during their translocation into mitochondria, from the channel to the import motor of the presequence translocase.

The TIM23 mitochondrial protein import complex: function and dysfunction.

Mitochondria acquire the majority of their proteins from the cytosol in a process that is mediated by intricate multimeric machineries designed to allow proteins to cross and/or to insert themselves into the two mitochondrial membranes. Ongoing studies carried out in yeast over the past few decades have led to the discovery of numerous protein components that constitute several mitochondrial translocases. One of these complexes, the mitochondrial TIM23, is the major translocase for matrix proteins and is the focus of this review. The components of the TIM23 complex are categorized into four functional types. The first type plays the role of receptor for preproteins in the intermembrane space. The second type forms the actual channel that allows proteins to cross the inner mitochondrial membrane. The third species functions as part of the motor that mediates the final steps of import across the inner membrane. Additional components play regulatory roles orchestrating the action of this myriad of subunits. Recent studies provide new insights into the function of the mammalian TIM23 complex and the role that it plays under pathological conditions.

GxxxG motifs hold the TIM23 complex together.

Approximately 99% of the mitochondrial proteome is nucleus-encoded, synthesized in the cytosol, and subsequently imported into and sorted to the correct compartment in the organelle. The translocase of the inner mitochondrial membrane 23 (TIM23) complex is the major protein translocase of the inner membrane, and is responsible for translocation of proteins across the inner membrane and their insertion into the inner membrane. Tim23 is the central component of the complex that forms the import channel. A high-resolution structure of the import channel is still missing, and structural elements important for its function are unknown. In the present study, we analyzed the importance of the highly abundant GxxxG motifs in the transmembrane segments of Tim23 for the structural integrity of the TIM23 complex. Of 10 glycines present in the GxxxG motifs in the first, second and third transmembrane segments of Tim23, mutations of three of them in transmembrane segments 1 and 2 resulted in a lethal phenotype, and mutations of three others in a temperature-sensitive phenotype. The remaining four caused no obvious growth phenotype. Importantly, none of the mutations impaired the import and membrane integration of Tim23 precursor into mitochondria. However, the severity of growth impairment correlated with the destabilization of the TIM23 complex. We conclude that the GxxxG motifs found in the first and second transmembrane segments of Tim23 are necessary for the structural integrity of the TIM23 complex.

Direct interaction of mitochondrial targeting presequences with purified components of the TIM23 protein complex.

Precursor proteins that are imported from the cytosol into the matrix of mitochondria carry positively charged amphipathic presequences and cross the inner membrane with the help of vital components of the TIM23 complex. It is currently unclear which subunits of the TIM23 complex recognize and directly bind to presequences. Here we analyzed the binding of presequence peptides to purified components of the TIM23 complex. The interaction of three different presequences with purified soluble domains of yeast Tim50 (Tim50IMS), Tim23 (Tim23IMS), and full-length Tim44 was examined. Using chemical cross-linking and surface plasmon resonance we demonstrate, for the first time, the ability of purified Tim50IMS and Tim44 to interact directly with the yeast Hsp60 presequence. We also analyzed their interaction with presequences derived from precursors of yeast mitochondrial 70-kDa heat shock protein (mHsp70) and of bovine cytochrome P450SCC. Moreover, we characterized the nature of the interactions and determined their KDs. On the basis of our results, we suggest a mechanism of translocation where stronger interactions of the presequences on the trans side of the channel support the import of precursor proteins through TIM23 into the matrix.

Chloroplast ß chaperonins from A. thaliana function with endogenous cpn10 homologs in vitro.

The involvement of type I chaperonins in bacterial and organellar protein folding has been well-documented. In E. coli and mitochondria, these ubiquitous and highly conserved proteins form chaperonin oligomers of identical 60 kDa subunits (cpn60), while in chloroplasts, two distinct cpn60 α and ß subunit types co-exist together. The primary sequence of α and ß subunits is ~50% identical, similar to their respective homologies to the bacterial GroEL. Moreover, the A. thaliana genome contains two α and four ß genes. The functional significance of this variability in plant chaperonin proteins has not yet been elucidated. In order to gain insight into the functional variety of the chloroplast chaperonin family members, we reconstituted ß homo-oligomers from A. thaliana following their expression in bacteria and subjected them to a structure-function analysis. Our results show for the first time, that A. thaliana ß homo-oligomers can function in vitro with authentic chloroplast co-chaperonins (ch-cpn10 and ch-cpn20). We also show that oligomers made up of different ß subunit types have unique properties and different preferences for co-chaperonin partners. We propose that chloroplasts may contain active ß homo-oligomers in addition to hetero-oligomers, possibly reflecting a variety of cellular roles.

Interaction of the Tim44 C-terminal domain with negatively charged phospholipids.

The translocation of proteins from the cytosol into the mitochondrial matrix is mediated by the coordinated action of the TOM complex in the outer membrane, as well as the TIM23 complex and its associated protein import motor in the inner membrane. The focus of this work is the peripheral inner membrane protein Tim44. Tim44 is a vital component of the mitochondrial protein translocation motor that anchors components of the motor to the TIM23 complex. For this purpose, Tim44 associates with the import channel by direct interaction with the Tim23 protein. Additionally, it was shown in vitro that Tim44 associates with acidic model membranes, in particular those containing cardiolipin. The latter interaction was shown to be mediated by the carboxy-terminal domain of Tim44 [Weiss, C., et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 8890-8894]. The aim of this study was to determine the precise recognition site for negative lipids in the C-terminal domain of Tim44. In particular, we wanted to examine the recently suggested hypothesis that acidic phospholipids associate with Tim44 via a hydrophobic cavity that is observed in the high-resolution structure of the C-terminal domain of the protein [Josyula, R., et al. (2006) J. Mol. Biol. 359, 798-804]. Molecular dynamics simulations suggest that (i) the hydrophobic tail of lipids may interact with Tim44 via the latter's hydrophobic cavity and (ii) a region, located in the N-terminal alpha-helix of the C-terminal domain (helices A1 and A2), may serve as a membrane attachment site. To validate this assumption, N-terminal truncations of yeast Tim44 were examined for their ability to bind cardiolipin-containing phospholipid vesicles. The results indicate that removal of the N-terminal alpha-helix (helix A1) abolishes the capacity of Tim44 to associate with cardiolipin-containing liposomes. We suggest that helices A1 and A2, in Tim44, jointly promote the association of the protein with acidic phospholipids.

Role of Tim50 in the transfer of precursor proteins from the outer to the inner membrane of mitochondria.

Transport of essentially all matrix and a number of inner membrane proteins is governed, entirely or in part, by N-terminal presequences and requires a coordinated action of the translocases of outer and inner mitochondrial membranes (TOM and TIM23 complexes). Here, we have analyzed Tim50, a subunit of the TIM23 complex that is implicated in transfer of precursors from TOM to TIM23. Tim50 is recruited to the TIM23 complex via Tim23 in an interaction that is essentially independent of the rest of the translocase. We find Tim50 in close proximity to the intermembrane space side of the TOM complex where it recognizes both types of TIM23 substrates, those that are to be transported into the matrix and those destined to the inner membrane, suggesting that Tim50 recognizes presequences. This function of Tim50 depends on its association with TIM23. We conclude that the efficient transfer of precursors between TOM and TIM23 complexes requires the concerted action of Tim50 with Tim23.

Interaction of Tim23 with Tim50 Is essential for protein translocation by the mitochondrial TIM23 complex.

The TIM23 complex is the major translocase of the mitochondrial inner membrane responsible for the import of essentially all matrix proteins and a number of inner membrane proteins. Tim23 and Tim50, two essential proteins of the complex, expose conserved domains into the intermembrane space that interact with each other. Here, we describe in vitro reconstitution of this interaction using recombinantly expressed and purified intermembrane space domains of Tim50 and Tim23. We established two independent methods, chemical cross-linking and surface plasmon resonance, to track their interaction. In addition, we identified mutations in Tim23 that abolish its interaction with Tim50 in vitro. These mutations also destabilized the interaction between the two proteins in vivo, leading to defective import of preproteins via the TIM23 complex and to cell death at higher temperatures. This is the first study to describe the reconstitution of the Tim50-Tim23 interaction in vitro and to identify specific residues of Tim23 that are vital for the interaction with Tim50.