Peter J. T. Dekker

The Tim core complex defines the number of mitochondrial translocation contact sites and can hold arrested preproteins in the absence of matrix Hsp70–Tim44

Preprotein import into mitochondria is mediated by translocases located in the outer and inner membranes (Tom and Tim) and a matrix Hsp70–Tim44 driving system. By blue native electrophoresis, we identify an ∼90K complex with assembled Tim23 and Tim17 as the core of the inner membrane import site for presequence‐containing preproteins. Preproteins spanning the two membranes link virtually all Tim core complexes with one in four Tom complexes in a stable 600K supercomplex. Neither mtHsp70 nor Tim44 are present in stoichiometric amounts in the 600K complex. Preproteins in transit stabilize the Tim core complex, preventing an exchange of subunits. Our studies define a central role for the Tim core complexes in mitochondrial protein import; they are not passive diffusion channels, but can stably interact with preproteins and determine the number of translocation contact sites. We propose the hypothesis that mtHsp70 functions in protein import not only by direct interaction with preproteins, but also by exerting a regulatory effect on the Tim channel.

Tom5 functionally links mitochondrial preprotein receptors to the general import pore

Most mitochondrial proteins are synthesized as preproteins on cytosolic polysomes and are subsequently imported into the organelle. The mitochondrial outer membrane contains a multisubunit preprotein translocase (Tom) which has receptors on the cytosolic side and a general import pore (GIP) in the membrane. Tom20–Tom22 and Tom70–Tom37 function as import receptors with a preference for preproteins that have amino-terminal presequences or internal targeting information, respectively. Tom40 is an essential constituent of the GIP,, whereas Tom6 and Tom7 modulate the assembly and dissociation of the Tom machinery,. Here we report the identification of Tom5, a small subunit that has a crucial role importing preproteins destined for all four mitochondrial subcompartments. Tom5 has a single membrane anchor and a cytosolic segment with a negative net charge, and accepts preproteins from the receptors and mediates their insertion into the GIP. We conclude that Tom5 represents a functional link between surface receptors and GIP, and is part of an ‘acid chain’ that guides the stepwise transport of positively charged mitochondrial targeting sequences.

Tom22 is a multifunctional organizer of the mitochondrial preprotein translocase

Mitochondrial preproteins are imported by a multisubunit translocase of the outer membrane (TOM), including receptor proteins and a general import pore. The central receptor Tom22 binds preproteins through both its cytosolic domain and its intermembrane space domain and is stably associated with the channel protein Tom40 (refs 11,12,13). Here we report the unexpected observation that a yeast strain can survive without Tom22, although it is strongly reduced in growth and the import of mitochondrial proteins. Tom22 is a multifunctional protein that is required for the higher-level organization of the TOM machinery. In the absence of Tom22, the translocase dissociates into core complexes, representing the basic import units, but lacks a tight control of channel gating. The single membrane anchor of Tom22 is required for a stable interaction between the core complexes, whereas its cytosolic domain serves as docking point for the peripheral receptors Tom20 and Tom70. Thus a preprotein translocase can combine receptor functions with distinct organizing roles in a multidomain protein.

Preprotein Translocase of the Outer Mitochondrial Membrane: Molecular Dissection and Assembly of the General Import Pore Complex

ABSTRACT The preprotein translocase of the outer mitochondrial membrane (Tom) is a multisubunit machinery containing receptors and a general import pore (GIP). We have analyzed the molecular architecture of the Tom machinery. The receptor Tom22 stably associates with Tom40, the main component of the GIP, in a complex with a molecular weight of ∼400,000 (∼400K), while the other receptors, Tom20 and Tom70, are more loosely associated with this GIP complex and can be found in distinct subcomplexes. A yeast mutant lacking both Tom20 and Tom70 can still form the GIP complex when sufficient amounts of Tom22 are synthesized. Besides the essential proteins Tom22 and Tom40, the GIP complex contains three small subunits, Tom5, Tom6, and Tom7. In mutant mitochondria lacking Tom6, the interaction between Tom22 and Tom40 is destabilized, leading to the dissociation of Tom22 and the generation of a subcomplex of ∼100K containing Tom40, Tom7, and Tom5. Tom6 is required to promote but not to maintain a stable association between Tom22 and Tom40. The following conclusions are suggested. (i) The GIP complex, containing Tom40, Tom22, and three small Tom proteins, forms the central unit of the outer membrane import machinery. (ii) Tom20 and Tom70 are not essential for the generation of the GIP complex. (iii) Tom6 functions as an assembly factor for Tom22, promoting its stable association with Tom40.

The essential yeast protein MIM44 (encoded by MPI1) is involved in an early step of preprotein translocation across the mitochondrial inner membrane

The essential yeast gene MPI1 encodes a mitochondrial membrane protein that is possibly involved in protein import into the organelle (A. C. Maarse, J. Blom, L. A. Grivell, and M. Meijer, EMBO J. 11:3619-3628, 1992). For this report, we determined the submitochondrial location of the MPI1 gene product and investigated whether it plays a direct role in the translocation of preproteins. By fractionation of mitochondria, the mature protein of 44 kDa was localized to the mitochondrial inner membrane and therefore termed MIM44. Import of the precursor of MIM44 required a membrane potential across the inner membrane and involved proteolytic processing of the precursor. A preprotein in transit across the mitochondrial membranes was cross-linked to MIM44, whereas preproteins arrested on the mitochondrial surface or fully imported proteins were not cross-linked. When preproteins were arrested at two distinct stages of translocation across the inner membrane, only preproteins at an early stage of translocation could be cross-linked to MIM44. Moreover, solubilized MIM44 was found to interact with in vitro-synthesized preproteins. We conclude that MIM44 is a component of the mitochondrial inner membrane import machinery and interacts with preproteins in an early step of translocation.

Multiple interactions of components mediating preprotein translocation across the inner mitochondrial membrane

The protein transport machinery of the inner mitochondrial membrane contains three essential Tim proteins. Tim17 and Tim23 are thought to build a preprotein translocation channel, while Tim44 transiently interacts with the matrix heat shock protein Hsp70 to form an ATP‐driven import motor. For this report we characterized the biogenesis and interactions of Tim proteins. (i) Import of the precursor of Tim44 into the inner membrane requires mtHsp70, whereas import and inner membrane integration of the precursors of Tim17 and Tim23 are independent of functional mtHsp70. (ii) Tim17 efficiently associates with Tim23 and mtHsp70, but only weakly with Tim44. (iii) Depletion of Tim44 does not affect the co‐precipitation of Tim17 with antibodies directed against mtHsp70. (iv) Tim23 associates with both Tim44 and Tim17, suggesting the presence of two Tim23 pools in the inner membrane, a Tim44–Tim23‐containing sub‐complex and a Tim23–Tim17‐containing sub‐complex. (v) The association of mtHsp70 with the Tim23–Tim17 sub‐complex is ATP sensitive and can be distinguished from the mtHsp70–Tim44 interaction by the differential influence of an amino acid substitution in mtHsp70. (vi) Genetic evidence, suppression of the protein import defect of a tim17 yeast mutant by overexpression of mtHsp70 and synthetic lethality of conditional mutants in the genes of Tim17 and mtHsp70, supports a functional interaction of mtHsp70 with Tim17. We conclude that the protein transport machinery of the mitochondrial inner membrane consists of dynamically interacting sub‐complexes, each of which transiently binds mtHsp70.

Protein Complexes Involved in Import of Mitochondrial Preproteins

Transport of preproteins across the mitochondrial outer and inner membrane involves the participation of separate protein complexes in both membranes. In recent years many subunits of the translocation channels have been characterized by biochemical and genetic studies in the yeast Saccharomyces cerevisiae (reviewed by Kubrich et al., 1995; Schatz and Dobberstein, 1996). In principle the different subunits can be functionally divided into four major groups: (i) the preprotein receptors, (ii) the translocation channel subunits of the outer membrane [together termed Tom proteins for “Translocase of the Outer Membrane”], (iii) the translocation channel(s) of the inner membrane and (iv) translocation motor subunits. The inner membrane proteins are termed Tim proteins for “Translocase of the Inner Membrane”. Contacts between individual subunits of the Tom or Tim complexes have been studied extensively by co-immune precipitation experiments after solubilization of mitochondria under nondenaturing conditions (generally with the detergent digitonin), or by in vivo crosslinking experiments (reviewed by Pfanner and Meijer, 1997). From these experiments the observer might have the impression that all proteins bind to all other proteins in the individual membranes, and all of them bind to translocating polypeptide chains. It should be emphasized, however, that these experiments do not make a distinction between stable structural interactions and interactions that are more transient and substoichiometric, but might still be functionally important. Furthermore, the relative abundance of many individual Tom and Tim proteins is unknown, making any prediction concerning the constitution of the membrane complexes unreliable.