Tim18p, a New Subunit of the TIM22 Complex That Mediates Insertion of Imported Proteins into the Yeast Mitochondrial Inner Membrane

ABSTRACT Import of carrier proteins from the cytoplasm into the mitochondrial inner membrane of yeast is mediated by a distinct system consisting of two soluble 70-kDa protein complexes in the intermembrane space and a 300-kDa complex in the inner membrane, the TIM22 complex. The TIM22 complex contains the peripheral subunits Tim9p, Tim10p, and Tim12p and the integral membrane subunits Tim22p and Tim54p. We identify here an additional subunit, an 18-kDa integral membrane protein termed Tim18p. This protein is made as a 21.9-kDa precursor which is imported into mitochondria and processed to its mature form. When mitochondria are gently solubilized, Tim18p comigrates with the other subunits of the TIM22 complex on nondenaturing gels and is coimmunoprecipitated with Tim54p and Tim12p. Tim18p does not cofractionate with the TIM23 complex upon immunoprecipitation or nondenaturing gel electrophoresis. Deletion of Tim18p decreases the growth rate of yeast cells by a factor of two and is synthetically lethal with temperature-sensitive mutations in Tim9p or Tim10p. It also impairs the import of several precursor proteins into isolated mitochondria, and lowers the apparent mass of the TIM22 complex. We suggest that Tim18p functions in the assembly and stabilization of the TIM22 complex but does not directly participate in protein insertion into the inner membrane.

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YME1L controls the accumulation of respiratory chain subunits and is required for apoptotic resistance, cristae morphogenesis, and cell proliferation

Molecular Biology of the Cell ◽

10.1091/mbc.e11-08-0674 ◽

2012 ◽

Vol 23 (6) ◽

pp. 1010-1023 ◽

Cited By ~ 89

Author(s):

Lukas Stiburek ◽

Jana Cesnekova ◽

Olga Kostkova ◽

Daniela Fornuskova ◽

Kamila Vinsova ◽

...

Keyword(s):

Cell Proliferation ◽

Membrane Protein ◽

Respiratory Chain ◽

Integral Membrane Protein ◽

Intermembrane Space ◽

Wild Type ◽

Inner Membrane ◽

Mitochondrial Inner Membrane ◽

Human Orthologue ◽

Excessive Accumulation

Mitochondrial ATPases associated with diverse cellular activities (AAA) proteases are involved in the quality control and processing of inner-membrane proteins. Here we investigate the cellular activities of YME1L, the human orthologue of the Yme1 subunit of the yeast i‑AAA complex, using stable short hairpin RNA knockdown and expression experiments. Human YME1L is shown to be an integral membrane protein that exposes its carboxy-terminus to the intermembrane space and exists in several complexes of 600–1100 kDa. The stable knockdown of YME1L in human embryonic kidney 293 cells led to impaired cell proliferation and apoptotic resistance, altered cristae morphology, diminished rotenone-sensitive respiration, and increased susceptibility to mitochondrial membrane protein carbonylation. Depletion of YME1L led to excessive accumulation of nonassembled respiratory chain subunits (Ndufb6, ND1, and Cox4) in the inner membrane. This was due to a lack of YME1L proteolytic activity, since the excessive accumulation of subunits was reversed by overexpression of wild-type YME1L but not a proteolytically inactive YME1L variant. Similarly, the expression of wild-type YME1L restored the lamellar cristae morphology of YME1L-deficient mitochondria. Our results demonstrate the importance of mitochondrial inner-membrane proteostasis to both mitochondrial and cellular function and integrity and reveal a novel role for YME1L in the proteolytic regulation of respiratory chain biogenesis.

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Phosphatidylserine transport by Ups2–Mdm35 in respiration-active mitochondria

The Journal of Cell Biology ◽

10.1083/jcb.201601082 ◽

2016 ◽

Vol 214 (1) ◽

pp. 77-88 ◽

Cited By ~ 44

Author(s):

Non Miyata ◽

Yasunori Watanabe ◽

Yasushi Tamura ◽

Toshiya Endo ◽

Osamu Kuge

Keyword(s):

Yeast Cells ◽

Intermembrane Space ◽

Diauxic Shift ◽

Inner Membrane ◽

Mitochondrial Inner Membrane ◽

Mitochondrial Functions ◽

Phospholipid Transfer ◽

Metabolic Transition ◽

Mitochondrial Intermembrane Space

Phosphatidylethanolamine (PE) is an essential phospholipid for mitochondrial functions and is synthesized mainly by phosphatidylserine (PS) decarboxylase at the mitochondrial inner membrane. In Saccharomyces cerevisiae, PS is synthesized in the endoplasmic reticulum (ER), such that mitochondrial PE synthesis requires PS transport from the ER to the mitochondrial inner membrane. Here, we provide evidence that Ups2–Mdm35, a protein complex localized at the mitochondrial intermembrane space, mediates PS transport for PE synthesis in respiration-active mitochondria. UPS2- and MDM35-null mutations greatly attenuated conversion of PS to PE in yeast cells growing logarithmically under nonfermentable conditions, but not fermentable conditions. A recombinant Ups2–Mdm35 fusion protein exhibited phospholipid-transfer activity between liposomes in vitro. Furthermore, UPS2 expression was elevated under nonfermentable conditions and at the diauxic shift, the metabolic transition from glycolysis to oxidative phosphorylation. These results demonstrate that Ups2–Mdm35 functions as a PS transfer protein and enhances mitochondrial PE synthesis in response to the cellular metabolic state.

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Two Intermembrane Space Tim Complexes Interact with Different Domains of Tim23p during Its Import into Mitochondria

The Journal of Cell Biology ◽

10.1083/jcb.150.6.1271 ◽

2000 ◽

Vol 150 (6) ◽

pp. 1271-1282 ◽

Cited By ~ 66

Author(s):

Alison J. Davis ◽

Naresh B. Sepuri ◽

Jason Holder ◽

Arthur E. Johnson ◽

Robert E. Jensen

Keyword(s):

Protein Complexes ◽

Intermediate Stage ◽

Terminal Segment ◽

Mitochondrial Outer Membrane ◽

Cross Linking ◽

Intermembrane Space ◽

Cross Link ◽

Inner Membrane ◽

Mitochondrial Inner Membrane

Tim23p (translocase of the inner membrane) is an essential import component located in the mitochondrial inner membrane. To determine how the Tim23 protein itself is transported into mitochondria, we used chemical cross-linking to identify proteins adjacent to Tim23p during its biogenesis. In the absence of an inner membrane potential, Tim23p is translocated across the mitochondrial outer membrane, but not inserted into the inner membrane. At this intermediate stage, we find that Tim23p forms cross-linked products with two distinct protein complexes of the intermembrane space, Tim8p–Tim13p and Tim9p–Tim10p. Tim9p and Tim10p cross-link to the COOH-terminal domain of the Tim23 protein, which carries all of the targeting signals for Tim23p. Therefore, our results suggest that the Tim9p–Tim10p complex plays a key role in Tim23p import. In contrast, Tim8p and Tim13p cross-link to the hydrophilic NH2-terminal segment of Tim23p, which does not carry essential import information and, thus, the role of Tim8p–Tim13p is unclear. Tim23p contains two matrix-facing, positively charged loops that are essential for its insertion into the inner membrane. The positive charges are not required for interaction with the Tim9p–Tim10p complex, but are essential for cross-linking of Tim23p to components of the inner membrane insertion machinery, including Tim54p, Tim22p, and Tim12p.

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The Assembly Pathway of the Mitochondrial Carrier Translocase Involves Four Preprotein Translocases

Molecular and Cellular Biology ◽

10.1128/mcb.02216-07 ◽

2008 ◽

Vol 28 (13) ◽

pp. 4251-4260 ◽

Cited By ~ 44

Author(s):

Karina Wagner ◽

Natalia Gebert ◽

Bernard Guiard ◽

Katrin Brandner ◽

Kaye N. Truscott ◽

...

Keyword(s):

Protein Complexes ◽

Intermembrane Space ◽

Inner Membrane ◽

Mitochondrial Carrier ◽

Important Principle ◽

Highly Active ◽

Mitochondrial Inner Membrane ◽

Assembly Pathway ◽

Oligomeric Complex ◽

The Individual

ABSTRACT The mitochondrial inner membrane contains preprotein translocases that mediate insertion of hydrophobic proteins. Little is known about how the individual components of these inner membrane preprotein translocases combine to form multisubunit complexes. We have analyzed the assembly pathway of the three membrane-integral subunits Tim18, Tim22, and Tim54 of the twin-pore carrier translocase. Tim54 displayed the most complex pathway involving four preprotein translocases. The precursor is translocated across the intermembrane space in a supercomplex of outer and inner membrane translocases. The TIM10 complex, which translocates the precursor of Tim22 through the intermembrane space, functions in a new posttranslocational manner: in case of Tim54, it is required for the integration of Tim54 into the carrier translocase. Tim18, the function of which has been unknown so far, stimulates integration of Tim54 into the carrier translocase. We show that the carrier translocase is built via a modular process and that each subunit follows a different assembly route. Membrane insertion and assembly into the oligomeric complex are uncoupled for each precursor protein. We propose that the mitochondrial assembly machinery has adapted to the needs of each membrane-integral subunit and that the uncoupling of translocation and oligomerization is an important principle to ensure continuous import and assembly of protein complexes in a highly active membrane.

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Conserved substrate binding by chaperones in the bacterial periplasm and the mitochondrial intermembrane space

Biochemical Journal ◽

10.1042/bj20070877 ◽

2007 ◽

Vol 409 (2) ◽

pp. 377-387 ◽

Cited By ~ 29

Author(s):

Felicity H. Alcock ◽

J. Günter Grossmann ◽

Ian E. Gentle ◽

Vladimir A. Likić ◽

Trevor Lithgow ◽

...

Keyword(s):

Substrate Binding ◽

Binding Specificity ◽

Yeast Cells ◽

Mitochondrial Outer Membrane ◽

Intermembrane Space ◽

Hydrophobic Membrane ◽

Inner Membrane ◽

Mitochondrial Inner Membrane ◽

Mitochondrial Intermembrane Space ◽

Substrate Proteins

Mitochondria were derived from intracellular bacteria and the mitochondrial intermembrane space is topologically equivalent to the bacterial periplasm. Both compartments contain ATP-independent chaperones involved in the transport of hydrophobic membrane proteins. The mitochondrial TIM (translocase of the mitochondrial inner membrane) 10 complex and the periplasmic chaperone SurA were examined in terms of evolutionary relation, structural similarity, substrate binding specificity and their function in transporting polypeptides for insertion into membranes. The two chaperones are evolutionarily unrelated; structurally, they are also distinct both in their characteristics, as determined by SAXS (small-angle X-ray scattering), and in pairwise structural comparison using the distance matrix alignment (DALILite server). Despite their structural differences, SurA and the TIM10 complex share a common binding specificity in Pepscan assays of substrate proteins. Comprehensive analysis of the binding on a total of 1407 immobilized 13-mer peptides revealed that the TIM10 complex, like SurA, does not bind hydrophobic peptides generally, but that both chaperones display selectivity for peptides rich in aromatic residues and with net positive charge. This common binding specificity was not sufficient for SurA to completely replace TIM10 in yeast cells in vivo. In yeast cells lacking TIM10, when SurA is targeted to the intermembrane space of mitochondria, it binds translocating substrate proteins, but fails to completely transfer the substrate to the translocase in the mitochondrial inner membrane. We suggest that SurA was incapable of presenting substrates effectively to the primitive TOM (translocase of the mitochondrial outer membrane) and TIM complexes in early mitochondria, and was replaced by the more effective small Tim chaperone.

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Cooperation of translocase complexes in mitochondrial protein import

The Journal of Cell Biology ◽

10.1083/jcb.200708199 ◽

2007 ◽

Vol 179 (4) ◽

pp. 585-591 ◽

Cited By ~ 56

Author(s):

Stephan Kutik ◽

Bernard Guiard ◽

Helmut E. Meyer ◽

Nils Wiedemann ◽

Nikolaus Pfanner

Keyword(s):

Outer Membrane ◽

Protein Import ◽

Protein Complexes ◽

Mitochondrial Protein ◽

Mitochondrial Proteins ◽

Intermembrane Space ◽

Mitochondrial Protein Import ◽

Inner Membrane ◽

Precursor Proteins ◽

Preprotein Translocation

Most mitochondrial proteins are synthesized in the cytosol and imported into one of the four mitochondrial compartments: outer membrane, intermembrane space, inner membrane, and matrix. Each compartment contains protein complexes that interact with precursor proteins and promote their transport. These translocase complexes do not act as independent units but cooperate with each other and further membrane complexes in a dynamic manner. We propose that a regulated coupling of translocases is important for the coordination of preprotein translocation and efficient sorting to intramitochondrial compartments.

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Characterization of Mmp37p, aSaccharomyces cerevisiaeMitochondrial Matrix Protein with a Role in Mitochondrial Protein Import

Molecular Biology of the Cell ◽

10.1091/mbc.e06-04-0366 ◽

2006 ◽

Vol 17 (9) ◽

pp. 4051-4062 ◽

Cited By ~ 47

Author(s):

Michelle R. Gallas ◽

Mary K. Dienhart ◽

Rosemary A. Stuart ◽

Roy M. Long

Keyword(s):

Matrix Protein ◽

Protein Import ◽

Mitochondrial Protein ◽

Temperature Sensitive ◽

Inner Membrane ◽

Mitochondrial Inner Membrane ◽

Close Proximity ◽

The Matrix ◽

Targeting Signal

Many mitochondrial proteins are encoded by nuclear genes and after translation in the cytoplasm are imported via translocases in the outer and inner membranes, the TOM and TIM complexes, respectively. Here, we report the characterization of the mitochondrial protein, Mmp37p (YGR046w) and demonstrate its involvement in the process of protein import into mitochondria. Haploid cells deleted of MMP37 are viable but display a temperature-sensitive growth phenotype and are inviable in the absence of mitochondrial DNA. Mmp37p is located in the mitochondrial matrix where it is peripherally associated with the inner membrane. We show that Mmp37p has a role in the translocation of proteins across the mitochondrial inner membrane via the TIM23-PAM complex and further demonstrate that substrates containing a tightly folded domain in close proximity to their mitochondrial targeting sequences display a particular dependency on Mmp37p for mitochondrial import. Prior unfolding of the preprotein, or extension of the region between the targeting signal and the tightly folded domain, relieves their dependency for Mmp37p. Furthermore, evidence is presented to show that Mmp37 may affect the assembly state of the TIM23 complex. On the basis of these findings, we hypothesize that the presence of Mmp37p enhances the early stages of the TIM23 matrix import pathway to ensure engagement of incoming preproteins with the mtHsp70p/PAM complex, a step that is necessary to drive the unfolding and complete translocation of the preprotein into the matrix.

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Tim18p Is a New Component of the Tim54p-Tim22p Translocon in the Mitochondrial Inner Membrane

Molecular Biology of the Cell ◽

10.1091/mbc.11.1.103 ◽

2000 ◽

Vol 11 (1) ◽

pp. 103-116 ◽

Cited By ~ 109

Author(s):

Oliver Kerscher ◽

Naresh B. Sepuri ◽

Robert E. Jensen

Keyword(s):

Growth Defect ◽

Inner Membrane ◽

New Members ◽

Mitochondrial Inner Membrane ◽

Native Electrophoresis ◽

Multiple Copies ◽

New Gene ◽

Synthetically Lethal ◽

Blue Native Electrophoresis ◽

Blue Native

The mitochondrial inner membrane contains two separate translocons: one required for the translocation of matrix-targeted proteins (the Tim23p-Tim17p complex) and one for the insertion of polytopic proteins into the mitochondrial inner membrane (the Tim54p-Tim22p complex). To identify new members of the Tim54p-Tim22p complex, we screened for high-copy suppressors of the temperature-sensitivetim54-1 mutant. We identified a new gene,TIM18, that encodes an integral protein of the inner membrane. The following genetic and biochemical observations suggest that the Tim18 protein is part of the Tim54p-Tim22p complex in the inner membrane: multiple copies of TIM18 suppress thetim54-1 growth defect; thetim18::HIS3 disruption is synthetically lethal with tim54-1; Tim54p and Tim22p can be coimmune precipitated with the Tim18 protein; and Tim18p, along with Tim54p and Tim22p, is detected in an ∼300-kDa complex after blue native electrophoresis. We propose that Tim18p is a new component of the Tim54p-Tim22p machinery that facilitates insertion of polytopic proteins into the mitochondrial inner membrane.

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