Biomedical subjects

Bernard Guiard

Publications and source records attributed to Bernard Guiard.

30 records · Page 2Linked to original sources

PubMed2004

Sam35 of the mitochondrial protein sorting and assembly machinery is a peripheral outer membrane protein essential for cell viability.

The mitochondrial outer membrane contains two integral proteins essential for cell viability, Tom40 of the translocase of the outer membrane (TOM complex) and Sam50 of the sorting and assembly machinery (SAM complex). Here we report the identification of Sam35, the first peripheral mitochondrial outer membrane protein that is essential for cell viability. Sam35 (encoded by the Saccharomyces cerevisiae ORF YHR083w) is a novel subunit of the SAM complex and is crucial for the assembly pathway of outer membrane beta-barrel proteins, such as the precursors of Tom40 and porin. Sam35 is not required for the import of inner membrane or matrix targeted proteins. The presence of two essential proteins in the SAM complex, Sam35 and Sam50, indicates that it plays a central role in mitochondrial biogenesis.

Amino Acid Sequence↗

PubMed2004

Biogenesis of the protein import channel Tom40 of the mitochondrial outer membrane: intermembrane space components are involved in an early stage of the assembly pathway.

Tom40 forms the central channel of the preprotein translocase of the mitochondrial outer membrane (TOM complex). The precursor of Tom40 is encoded in the nucleus, synthesized in the cytosol, and imported into mitochondria via a multi-step assembly pathway that involves the mature TOM complex and the sorting and assembly machinery of the outer membrane (SAM complex). We report that opening of the mitochondrial intermembrane space by swelling blocks the assembly pathway of the beta-barrel protein Tom40. Mitochondria with defects in small Tim proteins of the intermembrane space are impaired in the Tom40 assembly pathway. Swelling as well as defects in the small Tim proteins inhibit an early stage of the Tom40 import pathway that is needed for formation of a Tom40-SAM intermediate. We propose that the biogenesis pathway of beta-barrel proteins of the outer mitochondrial membrane not only requires TOM and SAM components, but also involves components of the intermembrane space.

Intracellular Membranes↗

PubMed2004

Pam16 has an essential role in the mitochondrial protein import motor.

Mitochondrial preproteins destined for the matrix are translocated by two channel-forming transport machineries, the translocase of the outer membrane and the presequence translocase of the inner membrane. The presequence translocase-associated protein import motor (PAM) contains four essential subunits: the matrix heat shock protein 70 (mtHsp70) and its three cochaperones Mge1, Tim44 and Pam18. Here we report that the PAM contains a fifth essential subunit, Pam16 (encoded by Saccharomyces cerevisiae YJL104W), which is selectively required for preprotein translocation into the matrix, but not for protein insertion into the inner membrane. Pam16 interacts with Pam18 and is needed for the association of Pam18 with the presequence translocase and for formation of a mtHsp70-Tim44 complex. Thus, Pam16 is a newly identified type of motor subunit and is required to promote a functional PAM reaction cycle, thereby driving preprotein import into the matrix.

Carrier Proteins↗

PubMed2004

The mitochondrial morphology protein Mdm10 functions in assembly of the preprotein translocase of the outer membrane.

The biogenesis of mitochondrial outer membrane proteins involves the general translocase of the outer membrane (TOM complex) and the sorting and assembly machinery (SAM complex). The two known subunits of the SAM complex, Mas37 and Sam50, are required for assembly of the abundant outer membrane proteins porin and Tom40. We have identified an unexpected subunit of the SAM complex, Mdm10, which is involved in maintenance of mitochondrial morphology. Mitochondria lacking Mdm10 are selectively impaired in the final steps of the assembly pathway of Tom40, including the association of Tom40 with the receptor Tom22 and small Tom proteins, while the biogenesis of porin is not affected. Yeast mutants of TOM40, MAS37, and SAM50 also show aberrant mitochondrial morphology. We conclude that Mdm10 plays a specific role in the biogenesis of the TOM complex, indicating a connection between the mitochondrial protein assembly apparatus and the machinery for maintenance of mitochondrial morphology.

Cells, Cultured↗

PubMed2003

A J-protein is an essential subunit of the presequence translocase-associated protein import motor of mitochondria.

Transport of preproteins into the mitochondrial matrix is mediated by the presequence translocase-associated motor (PAM). Three essential subunits of the motor are known: mitochondrial Hsp70 (mtHsp70); the peripheral membrane protein Tim44; and the nucleotide exchange factor Mge1. We have identified the fourth essential subunit of the PAM, an essential inner membrane protein of 18 kD with a J-domain that stimulates the ATPase activity of mtHsp70. The novel J-protein (encoded by PAM18/YLR008c/TIM14) is required for the interaction of mtHsp70 with Tim44 and protein translocation into the matrix. We conclude that the reaction cycle of the PAM of mitochondria involves an essential J-protein.

Amino Acid Sequence↗

PubMed2003

The proteome of Saccharomyces cerevisiae mitochondria.

We performed a comprehensive approach to determine the proteome of Saccharomyces cerevisiae mitochondria. The proteins of highly pure yeast mitochondria were separated by several independent methods and analyzed by tandem MS. From >20 million MS spectra, 750 different proteins were identified, indicating an involvement of mitochondria in numerous cellular processes. All known components of the oxidative phosphorylation machinery, the tricarboxylic acid cycle, and the stable mitochondria-encoded proteins were found. Based on the mitochondrial proteins described in the literature so far, we calculate that the identified proteins represent approximately 90% of all mitochondrial proteins. The function of a quarter of the identified proteins is unknown. The mitochondrial proteome will provide an important database for the analysis of new mitochondrial and mitochondria-associated functions and the characterization of mitochondrial diseases.

Citric Acid Cycle↗

PubMed2003

An essential role of Sam50 in the protein sorting and assembly machinery of the mitochondrial outer membrane.

The preprotein translocase of the outer mitochondrial membrane (TOM complex) contains one essential subunit, the channel Tom40. The assembly pathway of the precursor of Tom40 involves the TOM complex and the sorting and assembly machinery (SAM complex) with the non-essential subunit Mas37. We have identified Sam50, the second essential protein of the mitochondrial outer membrane. Sam50 contains a beta-barrel domain conserved from bacteria to man and is a subunit of the SAM complex. Yeast mutants of Sam50 are defective in the assembly pathways of Tom40 and the abundant outer membrane protein porin, while the import of matrix proteins is not affected. Thus the protein sorting and assembly machinery of the mitochondrial outer membrane involves an essential, conserved protein.

Amino Acid Sequence↗

PubMed2003

Mitochondrial translocation contact sites: separation of dynamic and stabilizing elements in formation of a TOM-TIM-preprotein supercomplex.

Preproteins with N-terminal presequences are imported into mitochondria at translocation contact sites that include the translocase of the outer membrane (TOM complex) and the presequence translocase of the inner membrane (TIM23 complex). Little is known about the functional cooperation of these translocases. We have characterized translocation contact sites by a productive TOM-TIM-preprotein supercomplex to address the role of three translocase subunits that expose domains to the intermembrane space (IMS). The IMS domain of the receptor Tom22 is required for stabilization of the translocation contact site supercomplex. Surprisingly, the N-terminal segment of the channel Tim23, which tethers the TIM23 complex to the outer membrane, is dispensable for both protein import and generation of the TOM-TIM supercomplex. Tim50, with its large IMS domain, is crucial for generation but not for stabilization of the supercomplex. Thus, Tim50 functions as a dynamic factor and the IMS domain of Tom22 represents a stabilizing element in formation of a productive translocation contact site supercomplex.

Carrier Proteins↗

PubMed2003

Mitochondria use different mechanisms for transport of multispanning membrane proteins through the intermembrane space.

The mitochondrial inner membrane contains numerous multispanning integral proteins. The precursors of these hydrophobic proteins are synthesized in the cytosol and therefore have to cross the mitochondrial outer membrane and intermembrane space to reach the inner membrane. While the import pathways of noncleavable multispanning proteins, such as the metabolite carriers, have been characterized in detail by the generation of translocation intermediates, little is known about the mechanism by which cleavable preproteins of multispanning proteins, such as Oxa1, are transferred from the outer membrane to the inner membrane. We have identified a translocation intermediate of the Oxa1 preprotein in the translocase of the outer membrane (TOM) and found that there are differences from the import mechanisms of carrier proteins. The intermembrane space domain of the receptor Tom22 supports the stabilization of the Oxa1 intermediate. Transfer of the Oxa1 preprotein to the inner membrane is not affected by inactivation of the soluble TIM complexes. Both the inner membrane potential and matrix heat shock protein 70 are essential to release the preprotein from the TOM complex, suggesting a close functional cooperation of the TOM complex and the presequence translocase of the inner membrane. We conclude that mitochondria employ different mechanisms for translocation of multispanning proteins across the aqueous intermembrane space.

Electron Transport Complex IV↗

PubMed2002

The ClpB homolog Hsp78 is required for the efficient degradation of proteins in the mitochondrial matrix.

Molecular chaperones perform vital functions in mitochondrial protein import and folding. In yeast mitochondria, two members of the Clp/Hsp100 chaperone family, Hsp78 and Mcx1, have been identified as homologs of the bacterial proteins ClpB and ClpX, respectively. In this report we employed a novel quantitative assay system to assess the role of Hsp78 and Mcx1 in protein degradation within the matrix. Mitochondria were preloaded with large amounts of two purified recombinant reporter proteins exhibiting different folding stabilities. Proteolysis of the imported substrate proteins depended on the mitochondrial level of ATP and was mediated by the matrix protease Pim1/LON. Degradation rates were found to be independent of the folding stability of the reporter proteins. Mitochondria from hsp78Delta cells exhibited a significant defect in the degradation efficiency of both substrates even at low temperature whereas mcx1Delta mitochondria showed wild-type activity. The proteolysis defect in hsp78Delta mitochondria was independent from the aggregation behavior of the substrate proteins. We conclude that Hsp78 is a genuine component of the mitochondrial proteolysis system required for the efficient degradation of substrate proteins in the matrix.

ATP-Dependent Proteases↗

PubMed2002

Tim22, the essential core of the mitochondrial protein insertion complex, forms a voltage-activated and signal-gated channel.

The protein insertion complex of the mitochondrial inner membrane is crucial for import of the numerous multitopic membrane proteins with internal targeting signals. Little is known about the molecular mechanism of this complex, including whether it forms a real channel or merely acts as scaffold for protein insertion. We report the unexpected observation that Tim22 is the only essential membrane-integrated subunit of the complex. Reconstituted Tim22 forms a hydrophilic, high-conductance channel with distinct opening states and pore diameters. The channel is voltage-activated and specifically responds to an internal targeting signal, but not to presequences. Thus, a protein insertion complex can combine three essential functions, signal recognition, channel formation, and energy transduction, in one central component.

Carrier Proteins↗

PubMed2002

Mitochondrial import of the ADP/ATP carrier: the essential TIM complex of the intermembrane space is required for precursor release from the TOM complex.

The mitochondrial intermembrane space contains a protein complex essential for cell viability, the Tim9-Tim10 complex. This complex is required for the import of hydrophobic membrane proteins, such as the ADP/ATP carrier (AAC), into the inner membrane. Different views exist about the role played by the Tim9-Tim10 complex in translocation of the AAC precursor across the outer membrane. For this report we have generated a new tim10 yeast mutant that leads to a strong defect in AAC import into mitochondria. Thereby, for the first time, authentic AAC is stably arrested in the translocase complex of the outer membrane (TOM), as shown by antibody shift blue native electrophoresis. Surprisingly, AAC is still associated with the receptors Tom70 and Tom20 when the function of Tim10 is impaired. The nonessential Tim8-Tim13 complex of the intermembrane space is not involved in the transfer of AAC across the outer membrane. These results define a two-step mechanism for translocation of AAC across the outer membrane. The initial insertion of AAC into the import channel is independent of the function of Tim9-Tim10; however, completion of translocation across the outer membrane, including release from the TOM complex, requires a functional Tim9-Tim10 complex.

Intracellular Membranes↗