General Import Pore Research Articles

The transport machinery for the import of preproteins across the outer mitochondrial membrane

In order for proteins to be imported into subcellular compartments, they must first traverse the organellar membranes. In mitochondria, hydrophilic protein channels in both the outer and inner membranes serve such a purpose. Recently, the channel protein of the outer mitochondrial membrane was identified to be Tom40. Tom40 is found in a high molecular weight complex termed the general import pore (GIP) complex where it is tightly associated with the receptor protein Tom22 along with Tom7, Tom6 and Tom5. Tom7 and Tom6 seem to modulate the dynamics of the GIP complex while Tom5 is involved in preprotein transfer from receptors to Tom40. The receptor proteins Tom70 and Tom20 associate with this complex in a weaker manner where they are involved in the initial recognition of preproteins. This review focuses on the identification and characterisation of the transport machinery of the outer mitochondrial membrane and how they are involved in the co-ordination and regulation of events required for the translocation of preproteins into mitochondria.

The preprotein translocase of the outer mitochondrial membrane: receptors and a general import pore.

Cytosol-synthesized preproteins destined for the mitochondria are transported across the outer membrane by the translocase of the mitochondrial outer membrane (TOM complex). This dynamic transport machinery can be divided into receptors that recognize preprotein targeting signals and components of the general import pore complex that mediate preprotein transport across the outer membrane. This review focuses on recent studies dealing with the central questions regarding the pore-forming subunits, and architecture and gating of the translocation channel of the outer membrane.

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 11-13). 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.

Biogenesis of Tim proteins of the mitochondrial carrier import pathway: differential targeting mechanisms and crossing over with the main import pathway.

Two major routes of preprotein targeting into mitochondria are known. Preproteins carrying amino-terminal signals mainly use Tom20, the general import pore (GIP) complex and the Tim23-Tim17 complex. Preproteins with internal signals such as inner membrane carriers use Tom70, the GIP complex, and the special Tim pathway, involving small Tims of the intermembrane space and Tim22-Tim54 of the inner membrane. Little is known about the biogenesis and assembly of the Tim proteins of this carrier pathway. We report that import of the preprotein of Tim22 requires Tom20, although it uses the carrier Tim route. In contrast, the preprotein of Tim54 mainly uses Tom70, yet it follows the Tim23-Tim17 pathway. The positively charged amino-terminal region of Tim54 is required for membrane translocation but not for targeting to Tom70. In addition, we identify two novel homologues of the small Tim proteins and show that targeting of the small Tims follows a third new route where surface receptors are dispensable, yet Tom5 of the GIP complex is crucial. We conclude that the biogenesis of Tim proteins of the carrier pathway cannot be described by either one of the two major import routes, but involves new types of import pathways composed of various features of the hitherto known routes, including crossing over at the level of the GIP.

Functional Staging of ADP/ATP Carrier Translocation across the Outer Mitochondrial Membrane

The ADP/ATP carrier (AAC) is the major representative of the inner membrane carrier proteins of mitochondria that are synthesized without cleavable presequences. The characterization of the import pathway of AAC into mitochondria has mainly depended on an operational staging system. Here, we introduce two approaches for analyzing the import of AAC, blue native electrophoresis and folding-induced translocation arrest, that allow a functional staging of AAC transport across the outer membrane. (i) Blue native electrophoresis permits a direct monitoring of the receptor stage of AAC and its chase into mitochondria. Binding to this stage requires the receptor protein Tom70 but not Tom37 or Tom20. (ii) A fusion protein between AAC and dihydrofolate reductase can be selectively arrested in the general import pore complex of the outer membrane by ligand induced folding of the passenger protein. Cross-linking demonstrates that the arrested preprotein is in close contact not only with several receptors and Tim10 but also with the channel protein Tom40, providing the first direct evidence that cleavable preproteins and carrier preproteins interact with the same outer membrane channel. The staging system presented here permits a molecular dissection of AAC transport across the outer mitochondrial membrane, relates it to functional units of the translocases, and indicates a coordinated and successive cooperation of distinct translocase subcomplexes during transfer of the preprotein.

Human Mitochondrial Complex I in Health and Disease

The authors are very grateful to Prof. Rob Sengers and Prof. Frans Trijbels, who founded the Nijmegen Center for Mitochondrial Disorders >20 years ago and who supported us throughout this large-scale project. We cordially thank all the Ph.D. students (Jan Loeffen, Ralf Triepels, Markus Schuelke, Sandy Budde, and Marieke Coenen) and technicians (Roel Smeets, Carin Buskens, Antoon Janssen, and Frans van den Brand) for their enthusiasm and the work they performed in achieving this first part of our research program. The Prinses Beatrix Fonds, the Stichting voor Kinderen die willen maar niet kunnen, and the Fonds Beoefening Wetenschap of the Nijmegen Children's Hospital are gratefully acknowledged for their financial support.

Preprotein translocase of the outer mitochondrial membrane: molecular dissection and assembly of the general import pore complex.

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 approximately 400,000 ( approximately 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 approximately 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.

Tom40 forms the hydrophilic channel of the mitochondrial import pore for preproteins [see comment

The mitochondrial outer membrane contains machinery for the import of preproteins encoded by nuclear genes. Eight different Tom (translocase of outer membrane) proteins have been identified that function as receptors and/or are related to a hypothetical general import pore. Many mitochondrial membrane channel activities have been described, including one related to Tim23 of the inner-membrane protein-import system; however, the pore-forming subunit(s) of the Tom machinery have not been identified until now. Here we describe the expression and functional reconstitution of Tom40, an integral membrane protein with mainly beta-sheet structure. Tom40 forms a cation-selective high-conductance channel that specifically binds to and transports mitochondrial-targeting sequences added to the cis side of the membrane. We conclude that Tom40 is the pore-forming subunit of the mitochondrial general import pore and that it constitutes a hydrophilic, approximately 22 A wide channel for the import of preproteins.

MITOCHONDRIAL PREPROTEIN TRANSLOCASE

Mitochondria import most of their proteins from the cytosol. Dynamic protein complexes in the mitochondrial outer and inner membranes are responsible for the specific recognition and membrane translocation of preproteins. The preprotein translocase of the outer mitochondrial membrane contains several import receptors and a general import pore. The preprotein translocase of the inner membrane consists of a channel interacting with preproteins in transit and an import motor that includes the matrix heat shock protein Hsp70. Acidic patches of import components are thought to guide the import of positively charged signal sequences (acid chain hypothesis). Energy input is derived from the inner membrane potential and ATP. Proteins in the mitochondrial matrix are required for proteolytic processing and folding of imported proteins. The dynamic nature of the membrane translocase permits sorting of preproteins at distinct stages of the import pathway.

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.

Tom7 modulates the dynamics of the mitochondrial outer membrane translocase and plays a pathway-related role in protein import.

The preprotein translocase of the outer mitochondrial membrane is a multi-subunit complex with receptors and a general import pore. We report the molecular identification of Tom7, a small subunit of the translocase that behaves as an integral membrane protein. The deletion of TOM7 inhibited the mitochondrial import of the outer membrane protein porin, whereas the import of preproteins destined for the mitochondrial interior was impaired only slightly. However, protein import into the mitochondrial interior was strongly inhibited when it occurred in two steps: preprotein accumulation at the outer membrane in the absence of a membrane potential and subsequent further import after the re-establishment of a membrane potential. The delay of protein import into tom7delta mitochondria seemed to occur after the binding of preproteins to the outer membrane receptor sites. A lack of Tom7 stabilized the interaction between the receptors Tom20 and Tom22 and the import pore component Tom40. This indicated that Tom7 exerts a destabilizing effect on part of the outer membrane translocase, whereas Tom6 stabilizes the interaction between the receptors and the import pore. Synthetic growth defects of the double mutants tom7delta tom20delta and tom7delta tom6delta provided genetic evidence for the functional relationship of Tom7 with Tom20 and Tom6. These results suggest that (i) Tom7 plays a role in sorting and accumulation of the preproteins at the outer membrane, and (ii) Tom7 and Tom6 perform complementary functions in modulating the dynamics of the outer membrane translocase.