Abstract
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.
Highlights
Many mitochondrial preproteins are synthesized with cleavable amino-terminal targeting sequences, several groups of mitochondrial proteins, in particular membrane proteins, are synthesized without presequences [1,2,3,4]
The following stages were defined: stage I, ADP/ATP carrier (AAC) bound to cytosolic cofactors; stage II, AAC bound to the mitochondrial surface; stage III, AAC was largely protected against externally added protease despite a dissipation of the inner membrane ⌬; stage IV, AAC was inserted into the inner membrane; stage V, AAC was dimeric and mature
Import Stages of ADP/ATP Carrier Dissected by Blue Native Electrophoresis—The preprotein of AAC was synthesized in vitro in rabbit reticulocyte lysates in the presence of [35S]methionine/cysteine
Summary
Unclear whether the receptor Tom comes into direct contact with carrier preproteins or plays more of an indirect role in their import. In vitro binding studies with purified receptors demonstrated an interaction of carrier preproteins with Tom and Tom, but not with Tom22 [22]. A carboxyl-terminal fusion of AAC with dihydrofolate reductase (DHFR) yielded a protein that could be selectively and stably arrested in the GIP complex by methotrexate-induced folding of the DHFR domain. This arrested preprotein yielded the first evidence for a close proximity of AAC with Tom and Tom. We compare the previous operational staging with the new functional staging of AAC import, which permits a molecular dissection of carrier transport across the outer mitochondrial membrane
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