A J-protein is an essential subunit of the presequence translocase-associated protein import motor of mitochondria

Transport of preproteins into the mitochondrial matrix is mediated by the presequence translocase-associated motor (PAM). Three essential subunits of the motor are known: mitochondrial Hsp70 (mtHsp70); the peripheral membrane protein Tim44; and the nucleotide exchange factor Mge1. We have identified the fourth essential subunit of the PAM, an essential inner membrane protein of 18 kD with a J-domain that stimulates the ATPase activity of mtHsp70. The novel J-protein (encoded by PAM18/YLR008c/TIM14) is required for the interaction of mtHsp70 with Tim44 and protein translocation into the matrix. We conclude that the reaction cycle of the PAM of mitochondria involves an essential J-protein.     

Pam16 has an essential role in the mitochondrial protein import motor

Mitochondrial preproteins destined for the matrix are translocated by two channel-forming transport machineries, the translocase of the outer membrane and the presequence translocase of the inner membrane. The presequence translocase-associated protein import motor (PAM) contains four essential subunits: the matrix heat shock protein 70 (mtHsp70) and its three cochaperones Mge1, Tim44 and Pam18. Here we report that the PAM contains a fifth essential subunit, Pam16 (encoded by Saccharomyces cerevisiae YJL104W), which is selectively required for preprotein translocation into the matrix, but not for protein insertion into the inner membrane. Pam16 interacts with Pam18 and is needed for the association of Pam18 with the presequence translocase and for formation of a mtHsp70-Tim44 complex. Thus, Pam16 is a newly identified type of motor subunit and is required to promote a functional PAM reaction cycle, thereby driving preprotein import into the matrix.     

The protein import motor of mitochondria

Proteins that are destined for the matrix of mitochondria are transported into this organelle by two translocases: the TOM complex, which transports proteins across the outer mitochondrial membrane; and the TIM23 complex, which gets them through the inner mitochondrial membrane. Two models have been proposed to explain how this protein-import machinery works -- a targeted Brownian ratchet, in which random motion is translated into vectorial motion, or a 'power stroke', which is exerted by a component of the import machinery. Here, we review the data for and against each model.     

Mitochondrial protein import motor: differential role of Tim44 in the recruitment of Pam17 and J-complex to the presequence translocase

The presequence translocase of the mitochondrial inner membrane (TIM23 complex) mediates the import of preproteins with amino-terminal presequences. To drive matrix translocation the TIM23 complex recruits the presequence translocase-associated motor (PAM) with the matrix heat shock protein 70 (mtHsp70) as central subunit. Activity and localization of mtHsp70 are regulated by four membrane-associated cochaperones: the adaptor protein Tim44, the stimulatory J-complex Pam18/Pam16, and Pam17. It has been proposed that Tim44 serves as molecular platform to localize mtHsp70 and the J-complex at the TIM23 complex, but it is unknown how Pam17 interacts with the translocase. We generated conditional tim44 yeast mutants and selected a mutant allele, which differentially affects the association of PAM modules with TIM23. In tim44-804 mitochondria, the interaction of the J-complex with the TIM23 complex is impaired, whereas unexpectedly the binding of Pam17 is increased. Pam17 interacts with the channel protein Tim23, revealing a new interaction site between TIM23 and PAM. Thus, the motor PAM is composed of functional modules that bind to different sites of the translocase. We suggest that Tim44 is not simply a scaffold for binding of motor subunits but plays a differential role in the recruitment of PAM modules to the inner membrane translocase.     

Localization of a synthetic presequence that blocks protein import into mitochondria

In the accompanying paper (Glaser, S. M., and Cumsky, M. G. (1990) J. Biol. Chem. 265, 8808-8816) we demonstrated that pL4-(1-22), a synthetic peptide corresponding to the N-terminal 22 residues of the cytochrome c oxidase subunit IV presequence, blocked protein import into mitochondria. Import inhibition was reversible and occurred at a step subsequent to the initial recognition and binding of precursor proteins to the mitochondrial surface. In the present work we have studied the nature of the association between the peptide and mitochondria, as well as determined its intramitochondrial location. We found that pL4-(1-22) was imported into mitochondria in a manner that was dependent upon the delta psi and that the majority of the mitochondrially associated peptide was in the membrane fraction. Density gradient analysis of total membranes indicated that pL4-(1-22) cofractionated with the inner membrane, although the possibility that it was present in both membranes could not be ruled out. It appeared to be inserted within the bilayer since it could not be extracted with salts, chaotropic agents, or high pH. We observed a steady decrease in the amount of pL4-(1-22) found within peptide-treated mitochondria over time. Coincident with this decrease was an increase in the ability of those mitochondria to import and process precursor proteins, suggesting that the peptide was ultimately turned over. The results presented here correlate well with those of the accompanying paper. Together they suggest that pL4-(1-22) blocks import at the level of the mitochondrial membranes, although the exact nature of the import block is not yet clear.     

Interaction of the J-protein heterodimer Pam18/Pam16 of the mitochondrial import motor with the translocon of the inner membrane

Import of proteins across the inner mitochondrial membrane through the Tim23:Tim17 translocase requires the function of an essential import motor having mitochondrial 70-kDa heat-shock protein (mtHsp70) at its core. The heterodimer composed of Pam18, the J-protein partner of mtHsp70, and the related protein Pam16 is a critical component of this motor. We report that three interactions contribute to association of the heterodimer with the translocon: the N terminus of Pam16 with the matrix side of the translocon, the inner membrane space domain of Pam18 (Pam18(IMS)) with Tim17, and the direct interaction of the J-domain of Pam18 with the J-like domain of Pam16. Pam16 plays a major role in translocon association, as alterations affecting the stability of the Pam18:Pam16 heterodimer dramatically affect association of Pam18, but not Pam16, with the translocon. Suppressors of the growth defects caused by alterations in the N terminus of Pam16 were isolated and found to be due to mutations in a short segment of TIM44, the gene encoding the peripheral membrane protein that tethers mtHsp70 to the translocon. These data suggest a model in which Tim44 serves as a scaffold for precise positioning of mtHsp70 and its cochaperone Pam18 at the translocon.     

Reevaluation of the role of the Pam18:Pam16 interaction in translocation of proteins by the mitochondrial Hsp70-based import motor

The heat-shock protein 70 (Hsp70)-based import motor, associated with the translocon on the matrix side of the mitochondrial inner membrane, drives translocation of proteins via cycles of binding and release. Stimulation of Hsp70's ATPase activity by the translocon-associated J-protein Pam18 is critical for this process. Pam18 forms a heterodimer with the structurally related protein Pam16, via their J-type domains. This interaction has been proposed to perform a critical regulatory function, inhibiting the ATPase stimulatory activity of Pam18. Using biochemical and genetic assays, we tested this hypothesis by assessing the in vivo function of Pam18 variants having altered abilities to stimulate Hsp70's ATPase activity. The observed pattern of genetic interactions was opposite from that predicted if the heterodimer serves an inhibitory function; instead the pattern was consistent with that of mutations known to cause reduction in the stability of the heterodimer. Analysis of a previously uncharacterized region of Pam16 revealed its requirement for formation of an active Pam18:Pam16 complex able to stimulate Hsp70's ATPase activity. Together, our data are consistent with the idea that Pam18 and Pam16 form a stable heterodimer and that the critical role of the Pam18:Pam16 interaction is the physical tethering of Pam18 to the translocon via its interaction with Pam16.     

The presequence of fumarase is exposed to the cytosol during import into mitochondria

The majority of mitochondrial proteins can be imported into mitochondria following termination of their translation in the cytosol. Import of fumarase and several other proteins into mitochondria does not appear to occur post-translationally according to standard in vivo and in vitro assays. However, the nature of interaction between the translation and translocation apparatuses during import of these proteins is unknown. Therefore, a major question is whether the nascent chains of these proteins are exposed to the cytosol during import into mitochondria. We asked directly if the presequence of fumarase can be cleaved by externally added mitochondrial processing peptidase (MPP) during import, using an in vitro translation-translocation coupled reaction. The presequence of fumarase was cleaved by externally added MPP during import, indicating a lack of, or a loose physical connection between, the translation and translocation of this protein. Exchanging the authentic presequence of fumarase for that of the more efficient Su9-ATPase presequence reduced the exposure of fumarase precursors to externally added MPP en route to mitochondria. Therefore, exposure to cytosolic MPP is dependent on the presequence and not on the mature part of fumarase. On the other hand, following translation in the absence of mitochondria, the authentic fumarase presequence and that of Su9-ATPase become inaccessible to added MPP when attached to mature fumarase. Thus, folding of the mature portion of fumarase, which conceals the presequence, is the reason for its inability to be imported in classical post-translational assays. Another unique feature of fumarase is its distribution between the mitochondria and the cytosol. We show that in vivo the switch of the authentic presequence with that of Su9-ATPase caused more fumarase molecules to be localized to the mitochondria. A possible mechanism by which the cytosolic exposure, the targeting efficiency, and the subcellular distribution of fumarase are dictated by the presequence is discussed.     

Protein unfolding by mitochondria. The Hsp70 import motor

Protein unfolding is a key step in the import of some proteins into mitochondria and chloroplasts and in the degradation of regulatory proteins by ATP-dependent proteases. In contrast to protein folding, the reverse process has remained largely uninvestigated until now. This review discusses recent discoveries on the mechanism of protein unfolding during translocation into mitochondria. The mitochondria can actively unfold preproteins by unraveling them from the N-terminus. The central component of the mitochondrial import motor, the matrix heat shock protein 70, functions by both pulling and holding the preproteins.     

The presequence of Arabidopsis serine hydroxymethyltransferase SHM2 selectively prevents import into mesophyll mitochondria

Serine hydroxymethyltransferases (SHMs) are important enzymes of cellular one-carbon metabolism and are essential for the photorespiratory glycine-into-serine conversion in leaf mesophyll mitochondria. In Arabidopsis (Arabidopsis thaliana), SHM1 has been identified as the photorespiratory isozyme, but little is known about the very similar SHM2. Although the mitochondrial location of SHM2 can be predicted, some data suggest that this particular isozyme could be inactive or not targeted into mitochondria. We report that SHM2 is a functional mitochondrial SHM. In leaves, the presequence of SHM2 selectively hinders targeting of the enzyme into mesophyll mitochondria. For this reason, the enzyme is confined to the vascular tissue of wild-type Arabidopsis, likely the protoxylem and/or adjacent cells, where it occurs together with SHM1. The resulting exclusion of SHM2 from the photorespiratory environment of mesophyll mitochondria explains why this enzyme cannot substitute for SHM1 in photorespiratory metabolism. Unlike the individual shm1 and shm2 null mutants, which require CO(2)-enriched air to inhibit photorespiration (shm1) or do not show any visible impairment (shm2), double-null mutants cannot survive in CO(2)-enriched air. It seems that SHM1 and SHM2 operate in a redundant manner in one-carbon metabolism of nonphotorespiring cells with a high demand of one-carbon units; for example, during lignification of vascular cells. We hypothesize that yet unknown kinetic properties of SHM2 might render this enzyme unsuitable for the high-folate conditions of photorespiring mesophyll mitochondria.     

The protein import motor of mitochondria: unfolding and trapping of preproteins are distinct and separable functions of matrix Hsp70.

Mitochondrial heat shock protein 70 (mtHsp70) functions in unfolding, translocation, and folding of imported proteins. Controversial models of mtHsp70 action have been discussed: (1) physical trapping of preproteins is sufficient to explain the various mtHsp70 functions, and (2) unfolding of preproteins requires an active motor function of mtHsp70 ("pulling"). Intragenic suppressors of a mutant mtHsp70 separate two functions: a nonlethal folding defect caused by enhanced trapping of preproteins, and a conditionally lethal unfolding defect caused by an impaired interaction of mtHsp70 with the membrane anchor Tim44. Even enhanced trapping in wild-type mitochondria does not generate a pulling force. The motor function of mtHsp70 cannot be explained by passive trapping alone but includes an essential ATP-dependent interaction with Tim44 to generate a pulling force and unfold preproteins.     

Pam17 is required for architecture and translocation activity of the mitochondrial protein import motor

Import of mitochondrial matrix proteins involves the general translocase of the outer membrane and the presequence translocase of the inner membrane. The presequence translocase-associated motor (PAM) drives the completion of preprotein translocation into the matrix. Five subunits of PAM are known: the preprotein-binding matrix heat shock protein 70 (mtHsp70), the nucleotide exchange factor Mge1, Tim44 that directs mtHsp70 to the inner membrane, and the membrane-bound complex of Pam16-Pam18 that regulates the ATPase activity of mtHsp70. We have identified a sixth motor subunit. Pam17 (encoded by the open reading frame YKR065c) is anchored in the inner membrane and exposed to the matrix. Mitochondria lacking Pam17 are selectively impaired in the import of matrix proteins and the generation of an import-driving activity of PAM. Pam17 is required for formation of a stable complex between the cochaperones Pam16 and Pam18 and promotes the association of Pam16-Pam18 with the presequence translocase. Our findings suggest that Pam17 is required for the correct organization of the Pam16-Pam18 complex and thus contributes to regulation of mtHsp70 activity at the inner membrane translocation site.     

A yeast mitochondrial presequence functions as a signal for targeting to plant mitochondria in vivo.

To date, the presequence of the mitochondrial beta-subunit of ATPase from tobacco is the only signal sequence that has been shown to target a foreign protein into plant mitochondria in vivo. Here we report that the presequence of a yeast mitochondrial protein directs bacterial beta-glucuronidase (GUS) specifically into the mitochondrial compartment of transgenic tobacco plants. Fusions between the presequence of the mitochondrial tryptophanyl-tRNA-synthetase gene from yeast and the GUS gene have been introduced into tobacco plants and yeast cells. In both systems, proteins containing the complete yeast mitochondrial presequence are efficiently imported in the mitochondria. Measurements of GUS activity in different subcellular fractions indicate that there is no substantial misrouting of the chimeric proteins in plant cells. In vitro synthesized GUS fusion proteins have a higher molecular weight than those found inside yeast and tobacco mitochondria, suggesting a processing of the precursors during import. Interestingly, fusion proteins translocated across the mitochondrial membranes of tobacco have the same size as those that are imported into yeast mitochondria. We conclude that the processing enzyme in plant mitochondria may recognize a proximate or even the same cleavage site within the mitochondrial tryptophanyl-tRNA-synthetase presequence as the matrix protease from yeast.     

The protein import motor of mitochondria: a targeted molecular ratchet driving unfolding and translocation

Unfolding and import of preproteins into mitochondria are facilitated by a molecular motor in which heat shock protein 70 (Hsp70) in the matrix plays an essential role. Here we present two different experimental approaches to analyze mechanisms underlying this function of Hsp70. First, preproteins containing stretches of glutamic acid (polyE) or glycine (polyG) repeats in front of folded domains were imported into mitochondria. This occurred although Hsp70 cannot pull on these stretches to unfold the folded domains, since it does not bind to polyE and polyG. Secondly, preproteins containing titin immunoglobulin (Ig)-like domains were imported into mitochondria, despite the fact that forces of >200 pN are required to mechanically unfold these domains. Since molecular motors generate forces of approximately 5 pN, Hsp70 could not promote unfolding of the Ig-like domains by mechanical pulling. Our observations suggest that Hsp70 acts as an element of a Brownian ratchet, which mediates unfolding and translocation of preproteins across the mitochondrial membranes.     

The cleavable presequence is not essential for import and assembly of the phosphate carrier of mammalian mitochondria but enhances the specificity and efficiency of import.

The phosphate carrier (PiC) of mammalian mitochondria is synthesized with a cleavable presequence, in contrast to other members of the mitochondrial family of inner membrane carrier proteins. The precursor of PiC is efficiently imported, proteolytically processed, and correctly assembled in isolated mitochondria. Here we report that a presequence-deficient PiC was imported with an efficiency of about 50% as compared with the authentic precursor of PiC. This mature-sized PiC was correctly assembled, demonstrating that the presequence is not essential for the assembly pathway. We found the following functions for the PiC presequence. (i) The presequence by itself was able to target a passenger protein to mitochondria with a low efficiency, suggesting that the mammalian PiC contains multiple targeting signals, the more efficient one(s) present in the mature protein part. (ii) Deletion of the presequence allowed a more efficient heterologous import of mammalian PiC into mitochondria from Saccharomyces cerevisiae and Neurospora crassa, indicating an important role of the presequence in determining the specificity of PiC import. (iii) Import of the presequence-deficient PiC required a higher membrane potential across the inner membrane than that of the presequence-carrying form. Therefore, the presequence also enhances the translocation of PiC into the inner membrane.     

Mitochondrial protein import. Requirement of presequence elements and tom components for precursor binding to the TOM complex

Protein translocation across the outer mitochondrial membrane is mediated by the translocator called the TOM (translocase of the outer mitochondrial membrane) complex. The TOM complex possesses two presequence binding sites on the cytosolic side (the cis site) and on the intermembrane space side (the trans site). Here we analyzed the requirement of presequence elements and subunits of the TOM complex for presequence binding to the cis and trans sites of the TOM complex. The N-terminal 14 residues of the presequence of subunit 9 of F(0)-ATPase are required for binding to the trans site. The interaction between the presequence and the cis site is not sufficient to anchor the precursor protein to the TOM complex. Tom7 constitutes or is close to the trans site and has overlapping functions with the C-terminal intermembrane space domain of Tom22 in the mitochondrial protein import.     

The mitochondrial presequence translocase: an essential role of Tim50 in directing preproteins to the import channel

Mitochondrial proteins with N-terminal targeting signals are transported across the inner membrane via the presequence translocase, which consists of membrane-integrated channel proteins and the matrix Hsp70 import motor. It has not been known how preproteins are directed to the import channel. We have identified the essential protein Tim50, which exposes its major domain to the intermembrane space. Tim50 interacts with preproteins in transit and directs them to the channel protein Tim23. Inactivation of Tim50 strongly inhibits the import of preproteins with a classical matrix-targeting signal, while preproteins carrying an additional inner membrane-sorting signal do not strictly depend on Tim50. Thus, Tim50 is crucial for guiding the precursors of matrix proteins to their insertion site in the inner membrane.