Biomedical subjects

W Voos

Publications and source records attributed to W Voos.

28 records · Page 2Linked to original sources

PubMed1995

Mitochondrial GrpE modulates the function of matrix Hsp70 in translocation and maturation of preproteins.

Mitochondrial GrpE (Mge1p) is a mitochondrial cochaperone essential for viability of the yeast Saccharomyces cerevisiae. To study the role of Mge1p in the biogenesis of mitochondrial proteins, we isolated a conditional mutant allele of MGE1 which conferred a temperature-sensitive growth phenotype and led to the accumulation of mitochondrial preproteins after shifting of the cells to the restrictive temperature. The mutant Mge1 protein was impaired in its interaction with the matrix heat shock protein mt-Hsp70. The mutant mitochondria showed a delayed membrane translocation of preproteins, and the maturation of imported proteins was impaired, as evidenced by the retarded second proteolytic processing of a preprotein in the matrix. Moreover, the aggregation of imported proteins was decreased in the mutant mitochondria. The mutant Mge1p differentially modulated the interaction of mt-Hsp70 with preproteins compared with the wild type, resulting in decreased binding to preproteins in membrane transit and enhanced binding to fully imported proteins. We conclude that the interaction of Mge1p with mt-Hsp70 promotes the progress of the Hsp70 reaction cycle, which is essential for import and maturation of mitochondrial proteins.

Alleles↗

PubMed1994

Mitochondrial GrpE is present in a complex with hsp70 and preproteins in transit across membranes.

We characterized a 24-kDa protein associated with matrix hsp70 (mt-hsp70) of Neurospora crassa and Saccharomyces cerevisiae mitochondria. By using specific antibodies, the protein was identified as MGE, a mitochondrial homolog of the prokaryotic heat shock protein GrpE. MGE extracted from mitochondria was quantitatively bound to hsp70. It was efficiently released from hsp70 by the addition of Mg-ATP but not by nonhydrolyzable ATP analogs or high salt. A mutant mt-hsp70, which was impaired in release of bound precursor proteins, released MGE in an ATP-dependent manner, indicating that precursor proteins and MGE bind to different sites of hsp70. A preprotein accumulated in transit across the mitochondrial membranes was specifically coprecipitated by either antibodies directed against MGE or antibodies directed against mt-hsp70. The preprotein accumulated at the outer membrane was not coprecipitated by either antibody preparation. After being imported into the matrix, the preprotein could be coprecipitated only by antibodies against mt-hsp70. We propose that mt-hsp70 and MGE cooperate in membrane translocation of preproteins.

Ascomycota↗

PubMed1993

Targeting and translocation of the phosphate carrier/p32 to the inner membrane of yeast mitochondria.

We analyzed the submitochondrial location and biogenesis pathway of the phosphate carrier (PiC), also termed p32, of Saccharomyces cerevisiae mitochondria, PiC/p32 was found to behave as an integral membrane protein that cofractionated with the ADP/ATP carrier of the inner membrane. Import of the precursor of PiC/p32 required a membrane potential across the inner membrane, supporting its localization to the inner membrane. This makes it unlikely that the major function of PiC/p32 is that of an import receptor on the surface of the mitochondrial outer membrane. Furthermore, we found that both receptors MOM72 and MOM19 were involved in the import pathway of the precursor of PiC/p32 with MOM72 being responsible for the bulk of import. Yeast PiC/p32 is thus not only structurally homologous to the ADP/ATP carrier, but has a similar targeting mechanism and submitochondrial location, supporting its classification as a member of the inner membrane carrier family.

Antibodies, Monoclonal↗

PubMed1993

A dual role for mitochondrial heat shock protein 70 in membrane translocation of preproteins.

The role of mitochondrial 70-kD heat shock protein (mt-hsp70) in protein translocation across both the outer and inner mitochondrial membranes was studied using two temperature-sensitive yeast mutants. The degree of polypeptide translocation into the matrix of mutant mitochondria was analyzed using a matrix-targeted preprotein that was cleaved twice by the processing peptidase. A short amino-terminal segment of the preprotein (40-60 amino acids) was driven into the matrix by the membrane potential, independent of hsp70 function, allowing a single cleavage of the presequence. Artificial unfolding of the preprotein allowed complete translocation into the matrix in the case where mutant mt-hsp70 had detectable binding activity. However, in the mutant mitochondria in which binding to mt-hsp70 could not be detected the mature part of the preprotein was only translocated to the intermembrane space. We propose that mt-hsp70 fulfills a dual role in membrane translocation of preproteins. (a) Mt-hsp70 facilitates unfolding of the polypeptide chain for translocation across the mitochondrial membranes. (b) Binding of mt-hsp70 to the polypeptide chain is essential for driving the completion of transport of a matrix-targeted preprotein across the inner membrane. This second role is independent of the folding state of the preprotein, thus identifying mt-hsp70 as a genuine component of the inner membrane translocation machinery. Furthermore we determined the sites of the mutations and show that both a functional ATPase domain and ATP are needed for mt-hsp70 to bind to the polypeptide chain and drive its translocation into the matrix.

Amino Acid Sequence↗

PubMed1993

Presequence and mature part of preproteins strongly influence the dependence of mitochondrial protein import on heat shock protein 70 in the matrix.

To test the hypothesis that 70-kD mitochondrial heat shock protein (mt-hsp70) has a dual role in membrane translocation of preproteins we screened preproteins in an attempt to find examples which required either only the unfoldase or only the translocase function of mt-hsp70. We found that a series of fusion proteins containing amino-terminal portions of the intermembrane space protein cytochrome b2 (cyt. b2) fused to dihydrofolate reductase (DHFR) were differentially imported into mitochondria containing mutant hsp70s. A fusion protein between the amino-terminal 167 residues of the precursor of cyt. b2 and DHFR was efficiently transported into mitochondria independently of both hsp70 functions. When the length of the cyt. b2 portion was increased and included the heme binding domain, the fusion protein became dependent on the unfoldase function of mt-hsp70, presumably caused by a conformational restriction of the heme-bound preprotein. In the absence of heme the noncovalent heme binding domain in the longer fusion proteins no longer conferred a dependence on the unfoldase function. When the cyt. b2 portion of the fusion protein was less than 167 residues, its import was still independent of mt-hsp70 function; however, deletion of the intermembrane space sorting signal resulted in preproteins that ended up in the matrix of wild-type mitochondria and whose translocation was strictly dependent on the translocase function of mt-hsp70. These findings provide strong evidence for a dual role of mt-hsp70 in membrane translocation and indicate that preproteins with an intermembrane space sorting signal can be correctly imported even in mutants with severely impaired hsp70 function.

Biological Transport↗

PubMed1993

The essential yeast protein MIM44 (encoded by MPI1) is involved in an early step of preprotein translocation across the mitochondrial inner membrane.

The essential yeast gene MPI1 encodes a mitochondrial membrane protein that is possibly involved in protein import into the organelle (A. C. Maarse, J. Blom, L. A. Grivell, and M. Meijer, EMBO J. 11:3619-3628, 1992). For this report, we determined the submitochondrial location of the MPI1 gene product and investigated whether it plays a direct role in the translocation of preproteins. By fractionation of mitochondria, the mature protein of 44 kDa was localized to the mitochondrial inner membrane and therefore termed MIM44. Import of the precursor of MIM44 required a membrane potential across the inner membrane and involved proteolytic processing of the precursor. A preprotein in transit across the mitochondrial membranes was cross-linked to MIM44, whereas preproteins arrested on the mitochondrial surface or fully imported proteins were not cross-linked. When preproteins were arrested at two distinct stages of translocation across the inner membrane, only preproteins at an early stage of translocation could be cross-linked to MIM44. Moreover, solubilized MIM44 was found to interact with in vitro-synthesized preproteins. We conclude that MIM44 is a component of the mitochondrial inner membrane import machinery and interacts with preproteins in an early step of translocation.

Fungal Proteins↗

PubMed1990

Precursor proteins in transit through mitochondrial contact sites interact with hsp70 in the matrix.

We previously reported that hsp70 in the mitochondrial matrix (mt-hsp 70 = Ssclp) is required for import of precursor proteins destined for the matrix or intermembrane space. Here we show that mt-hsp70 is also needed for the import of mitochondrial inner membrane proteins. In particular, the precursor of ADP/ATP carrier that is known not to interact with hsp60 on its assembly pathway requires functional mt-hsp70 for import, suggesting a general role of mt-hsp70 in membrane translocation of precursors. Moreover, a precursor arrested in contact sites was specifically co-precipitated with antibodies directed against mt-hsp70. We conclude that mt-hsp70 is directly involved in the translocation of many, if not all, precursor proteins that are transported across the inner membrane.

Biological Transport↗

PubMed1987

Putative promoter elements for the ribosomal RNA genes of the thermoacidophilic archaebacterium Sulfolobus sp. strain B12.

In Sulfolobus sp. strain B12, single-copy genes encode the three ribosomal RNAs. The genes for the 16S rRNA and for the 23S rRNA are closely linked but separated from the 5S rRNA gene. Transcription of the 16S/23S rRNA gene cluster starts 139 nucleotides upstream of the 5'-end of mature 16S rRNA. For the 5S rRNA gene the point of transcription initiation coincides with the 5'-end of mature 5S rRNA. The comparison of the upstream regions for these transcriptional start sites shows the presence of a completely conserved trinucleotide sequence around the point of transcription initiation and a completely conserved octanucleotide sequence about 22 nucleotides upstream of it. These sequences are only moderately homologous to putative promoter elements for stable RNA genes in the closely related archaebacterium Thermoproteus tenax (1), but they are very similar to corresponding sequences in the distantly related archaebacterium Methanococcus vannielii (2). The consensus sequence found for Sulfolobus and Methanococcus could therefore constitute the archetype of an archaebacterial promoter for stable RNA genes.

Archaea↗

PubMedDate not supplied

Protein translocation into mitochondria.

The biogenesis of mitochondria requires the translocation of most mitochondrial proteins across two biological membranes. Mitochondrial preproteins are synthesized in the cytosol carrying targeting information, that is recognized by specific receptor proteins. The precursor polypeptides are transported across both mitochondrial membranes via three large integral membrane protein complexes forming specialized preprotein translocases. A soluble protein complex in the matrix provides the ATP-dependent translocation force, responsible for the movement and unfolding of the bulk polypeptide chain. After the removal of the targeting sequence, imported proteins fold into their native conformation with the help of chaperone proteins in the mitochondrial matrix.

Biological Transport, Active↗

PubMedDate not supplied

The mitochondrial protein import motor.

Mitochondrial proteins are synthesized as precursor proteins in the cytosol and are posttranslationally imported into the organelle. A complex system of translocation machineries recognizes and transports the precursor polypeptide across the mitochondrial membranes. Energy for the translocation process is mainly supplied by the mitochondrial membrane potential (deltapsi) and the hydrolysis of ATP. Mitochondrial Hsp70 (mtHsp70) has been identified as the major ATPase driving the membrane transport of the precursor polypeptides into the mitochondrial matrix. Together with the partner proteins Tim44 and Mge1, mtHsp70 forms an import motor complex interacting with the incoming preproteins at the inner face of the inner membrane. This import motor complex drives the movement of the polypeptides in the translocation channel and the unfolding of carboxy-terminal parts of the preproteins on the outside of the outer membrane. Two models of the molecular mechanism of mtHsp70 during polypeptide translocation are discussed. In the 'trapping' model, precursor movement is generated by Brownian movement of the polypeptide chain in the translocation pore. This random movement is made vectorial by the interaction with mtHsp70 in the matrix. The detailed characterization of conditional mutants of the import motor complex provides the basis for an extended model. In this 'pulling' model, the attachment of mtHsp70 at the inner membrane via Tim44 and a conformational change induced by ATP results in the generation of an inward-directed force on the bound precursor polypeptide. This active role of the import motor complex is necessary for the translocation of proteins containing tightly folded domains. We suggest that both mechanisms complement each other to reach a high efficiency of preprotein import.

Animals↗