Towards a molecular mechanism underlying mitochondrial protein import through the TOM and TIM23 complexes
Nearly all mitochondrial proteins need to be targeted for import from the cytosol. For the majority, the first port of call is the translocase of the outer membrane (TOM complex), followed by a procession of alternative molecular machines, conducting transport to their final destination. The pre-seq...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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eLife Sciences Publications Ltd
2022-06-01
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| Series: | eLife |
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| Online Access: | https://elifesciences.org/articles/75426 |
| _version_ | 1797998342991511552 |
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| author | Holly C Ford William J Allen Gonçalo C Pereira Xia Liu Mark Simon Dillingham Ian Collinson |
| author_facet | Holly C Ford William J Allen Gonçalo C Pereira Xia Liu Mark Simon Dillingham Ian Collinson |
| author_sort | Holly C Ford |
| collection | DOAJ |
| description | Nearly all mitochondrial proteins need to be targeted for import from the cytosol. For the majority, the first port of call is the translocase of the outer membrane (TOM complex), followed by a procession of alternative molecular machines, conducting transport to their final destination. The pre-sequence translocase of the inner membrane (TIM23-complex) imports proteins with cleavable pre-sequences. Progress in understanding these transport mechanisms has been hampered by the poor sensitivity and time resolution of import assays. However, with the development of an assay based on split NanoLuc luciferase, we can now explore this process in greater detail. Here, we apply this new methodology to understand how ∆ψ and ATP hydrolysis, the two main driving forces for import into the matrix, contribute to the transport of pre-sequence-containing precursors (PCPs) with varying properties. Notably, we found that two major rate-limiting steps define PCP import time: passage of PCP across the outer membrane and initiation of inner membrane transport by the pre-sequence – the rates of which are influenced by PCP size and net charge. The apparent distinction between transport through the two membranes (passage through TOM is substantially complete before PCP-TIM engagement) is in contrast with the current view that import occurs through TOM and TIM in a single continuous step. Our results also indicate that PCPs spend very little time in the TIM23 channel – presumably rapid success or failure of import is critical for maintenance of mitochondrial fitness. |
| first_indexed | 2024-04-11T10:47:08Z |
| format | Article |
| id | doaj.art-e4307bb25f824090b7a55e93b605833c |
| institution | Directory Open Access Journal |
| issn | 2050-084X |
| language | English |
| last_indexed | 2024-04-11T10:47:08Z |
| publishDate | 2022-06-01 |
| publisher | eLife Sciences Publications Ltd |
| record_format | Article |
| series | eLife |
| spelling | doaj.art-e4307bb25f824090b7a55e93b605833c2022-12-22T04:29:02ZengeLife Sciences Publications LtdeLife2050-084X2022-06-011110.7554/eLife.75426Towards a molecular mechanism underlying mitochondrial protein import through the TOM and TIM23 complexesHolly C Ford0https://orcid.org/0000-0003-1400-8049William J Allen1https://orcid.org/0000-0002-9513-4786Gonçalo C Pereira2https://orcid.org/0000-0001-9638-0615Xia Liu3Mark Simon Dillingham4https://orcid.org/0000-0002-4612-7141Ian Collinson5https://orcid.org/0000-0002-3931-0503School of Biochemistry, University of Bristol, Bristol, United KingdomSchool of Biochemistry, University of Bristol, Bristol, United KingdomSchool of Biochemistry, University of Bristol, Bristol, United KingdomSchool of Biochemistry, University of Bristol, Bristol, United KingdomSchool of Biochemistry, University of Bristol, Bristol, United KingdomSchool of Biochemistry, University of Bristol, Bristol, United KingdomNearly all mitochondrial proteins need to be targeted for import from the cytosol. For the majority, the first port of call is the translocase of the outer membrane (TOM complex), followed by a procession of alternative molecular machines, conducting transport to their final destination. The pre-sequence translocase of the inner membrane (TIM23-complex) imports proteins with cleavable pre-sequences. Progress in understanding these transport mechanisms has been hampered by the poor sensitivity and time resolution of import assays. However, with the development of an assay based on split NanoLuc luciferase, we can now explore this process in greater detail. Here, we apply this new methodology to understand how ∆ψ and ATP hydrolysis, the two main driving forces for import into the matrix, contribute to the transport of pre-sequence-containing precursors (PCPs) with varying properties. Notably, we found that two major rate-limiting steps define PCP import time: passage of PCP across the outer membrane and initiation of inner membrane transport by the pre-sequence – the rates of which are influenced by PCP size and net charge. The apparent distinction between transport through the two membranes (passage through TOM is substantially complete before PCP-TIM engagement) is in contrast with the current view that import occurs through TOM and TIM in a single continuous step. Our results also indicate that PCPs spend very little time in the TIM23 channel – presumably rapid success or failure of import is critical for maintenance of mitochondrial fitness.https://elifesciences.org/articles/75426mitochondriaimportkineticsprotein transport |
| spellingShingle | Holly C Ford William J Allen Gonçalo C Pereira Xia Liu Mark Simon Dillingham Ian Collinson Towards a molecular mechanism underlying mitochondrial protein import through the TOM and TIM23 complexes eLife mitochondria import kinetics protein transport |
| title | Towards a molecular mechanism underlying mitochondrial protein import through the TOM and TIM23 complexes |
| title_full | Towards a molecular mechanism underlying mitochondrial protein import through the TOM and TIM23 complexes |
| title_fullStr | Towards a molecular mechanism underlying mitochondrial protein import through the TOM and TIM23 complexes |
| title_full_unstemmed | Towards a molecular mechanism underlying mitochondrial protein import through the TOM and TIM23 complexes |
| title_short | Towards a molecular mechanism underlying mitochondrial protein import through the TOM and TIM23 complexes |
| title_sort | towards a molecular mechanism underlying mitochondrial protein import through the tom and tim23 complexes |
| topic | mitochondria import kinetics protein transport |
| url | https://elifesciences.org/articles/75426 |
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