Biomedical subjects

Agnieszka Chacinska

Publications and source records attributed to Agnieszka Chacinska.

18 recordsLinked to original sources

PubMed2006

The Tim21 binding domain connects the preprotein translocases of both mitochondrial membranes.

Proteins destined for the mitochondrial matrix are imported by the translocase of the outer membrane--the TOM complex--and the presequence translocase of the inner membrane--the TIM23 complex. At present, there is no structural information on components of the presequence translocase. Tim21, a subunit of the presequence translocase consisting of a membrane anchor and a carboxy-terminal domain exposed to the intermembrane space, directly connects the TOM and TIM23 complexes by binding to the intermembrane space domain of the Tom22 receptor. We crystallized the binding domain of Tim21 of Saccharomyces cerevisiae and determined its structure at 1.6 A resolution. The Tim21 structure represents a new alpha/beta-mixed protein fold with two alpha-helices flanked by an extended eight-stranded beta-sheet. We also identified a core sequence of Tom22 that binds to Tim21. Furthermore, negatively charged amino-acid residues of Tom22 are important for binding to Tim21. Here we suggest a mechanism for the TOM-TIM interaction.

Amino Acid Sequence↗

PubMed2006

Novel mitochondrial intermembrane space proteins as substrates of the MIA import pathway.

Mitochondria consist of four compartments, the outer membrane, intermembrane space (IMS), inner membrane and the matrix. Most mitochondrial proteins are synthesized as precursors in the cytosol and have to be imported into these compartments. While the protein import machineries of the outer membrane, inner membrane and matrix have been investigated in detail, a specific mitochondrial machinery for import and assembly of IMS proteins, termed MIA, was identified only recently. To date, only a very small number of substrate proteins of the MIA pathway have been identified. The substrates contain characteristic cysteine motifs, either a twin Cx(3)C or a twin Cx(9)C motif. The largest MIA substrates known possess a molecular mass of 11 kDa, implying that this new import pathway has a very small size limit. Here, we have compiled a list of Saccharomyces cerevisiae proteins with a twin Cx(9)C motif and identified three IMS proteins that were previously localized to incorrect cellular compartments by tagging approaches. Mdm35, Mic14 (YDR031w) and Mic17 (YMR002w) require the two essential subunits, Mia40 and Erv1, of the MIA machinery for their localization in the mitochondrial IMS. With a molecular mass of 14 kDa and 17 kDa, respectively, Mic14 and Mic17 are larger than the known MIA substrates. Remarkably, the precursor of Erv1 itself is imported via the MIA pathway. As Erv1 has a molecular mass of 22 kDa and a twin Cx(2)C motif, this study demonstrates that the MIA pathway can transport substrates that are twice as large as the substrates known to date and is not limited to proteins with twin Cx(3)C or Cx(9)C motifs. However, tagging of MIA substrates can interfere with their subcellular localization, indicating that the proper localization of mitochondrial IMS proteins requires the characterization of the authentic untagged proteins.

Amino Acid Motifs↗

PubMed2006

Tim50 maintains the permeability barrier of the mitochondrial inner membrane.

Transport of metabolites across the mitochondrial inner membrane is highly selective, thereby maintaining the electrochemical proton gradient that functions as the main driving force for cellular adenosine triphosphate synthesis. Mitochondria import many preproteins via the presequence translocase of the inner membrane. However, the reconstituted Tim23 protein constitutes a pore remaining mainly in its open form, a state that would be deleterious in organello. We found that the intermembrane space domain of Tim50 induced the Tim23 channel to close. Presequences overcame this effect and activated the channel for translocation. Thus, the hydrophilic cis domain of Tim50 maintains the permeability barrier of mitochondria by closing the translocation pore in a presequence-regulated manner.

Cell Membrane Permeability↗

PubMed2006

Distinct yet linked: chaperone networks in the eukaryotic cytosol.

The terms chaperone and heat-shock protein are frequently used as synonyms, but this is an oversimplification. Although one subset of chaperones is induced by heat stress, a distinct group fails to respond in the same manner. Recent work reveals that this latter group is linked to the translational apparatus and functions in co-translational processes.

Animals↗

PubMed2006

Chaperoning through the mitochondrial intermembrane space.

The first high-resolution structure of a mitochondrial translocase complex, the Tim9-Tim10 chaperone, is reported by Webb et al. (2006) in a recent issue of Molecular Cell, providing important insight in the transport of hydrophobic proteins through the aqueous intermembrane space and the mechanisms of protein assembly.

Escherichia coli Proteins↗

PubMed2005

The essential mitochondrial protein Erv1 cooperates with Mia40 in biogenesis of intermembrane space proteins.

The proteins of the mitochondrial intermembrane space (IMS) are encoded by nuclear genes and synthesized on cytosolic ribosomes. While some IMS proteins are imported by the classical presequence pathway that involves the membrane potential deltapsi across the inner mitochondrial membrane and proteolytic processing to release the mature protein to the IMS, the import of numerous small IMS proteins is independent of a deltapsi and does not include proteolytic processing. The biogenesis of small IMS proteins requires an essential mitochondrial IMS import and assembly protein, termed Mia40. Here, we show that Erv1, a further essential IMS protein that has been reported to function as a sulfhydryl oxidase and participate in biogenesis of Fe/S proteins, is also required for the biogenesis of small IMS proteins. We generated a temperature-sensitive yeast mutant of Erv1 and observed a strong reduction of the levels of small IMS proteins upon shift of the cells to non-permissive temperature. Isolated erv1-2 mitochondria were selectively impaired in import of small IMS proteins while protein import pathways to other mitochondrial subcompartments were not affected. Small IMS precursor proteins remained associated with Mia40 in erv1-2 mitochondria and were not assembled into mature oligomeric complexes. Moreover, Erv1 associated with Mia40 in a reductant-sensitive manner. We conclude that two essential proteins, Mia40 and Erv1, cooperate in the assembly pathway of small proteins of the mitochondrial IMS.

Mitochondrial Membrane Transport Proteins↗

PubMed2005

Inactivation of the mitochondrial heat shock protein zim17 leads to aggregation of matrix hsp70s followed by pleiotropic effects on morphology and protein biogenesis.

The biogenesis of mitochondrial matrix proteins involves the translocase of the outer membrane, the presequence translocase of the inner membrane and the presequence translocase-associated motor. The mitochondrial heat shock protein 70 (mtHsp70) forms the central core of the motor. Recent studies led to the identification of Zim17, a mitochondrial zinc finger motif protein that interacts with mtHsp70. Different views have been reported on the localization of Zim17 in the mitochondrial inner membrane or matrix. Depletion of Zim17 impairs several critical mitochondrial processes, leading to inhibition of protein import, defects of Fe/S protein biogenesis and aggregation of Hsp70s in the matrix. Additionally, we found that inactivation of Zim17 altered the morphology of mitochondria. These pleiotropic effects raise the question of the specific function of Zim17 in mitochondria. Here, we report that Zim17 is a heat shock protein of the mitochondrial matrix that is loosely associated with the inner membrane. To address the function of Zim17 in organello, we generated a temperature-sensitive mutant allele of the ZIM17 gene in yeast. Upon a short-term shift of the yeast mutant cells to a non-permissive temperature, matrix Hsp70s aggregated while protein import, Fe/S protein activity and mitochondrial morphology were not, or only mildly, affected. Only after a long-term shift to non-permissive temperature, were strong defects in protein import, Fe/S protein activity and mitochondrial morphology observed. These findings suggest that the heat shock protein Zim17 plays a specific role in preventing protein aggregation in the mitochondrial matrix, and that aggregation of Hsp70s causes pleiotropic effects on protein biogenesis and mitochondrial morphology.

Dimerization↗

PubMed2005

Mitochondrial presequence translocase: switching between TOM tethering and motor recruitment involves Tim21 and Tim17.

The presequence translocase of the inner mitochondrial membrane (TIM23 complex) operates at a central junction of protein import. It accepts preproteins from the outer membrane TOM complex and directs them to inner membrane insertion or, in cooperation with the presequence translocase-associated motor (PAM), to the matrix. Little is known of how the TIM23 complex coordinates these tasks. We have identified Tim21 (YGR033c) that interacts with the TOM complex. Tim21 is specific for a TIM23 form that cooperates with TOM and promotes inner membrane insertion. Protein translocation into the matrix requires a switch to a Tim21-free, PAM bound presequence translocase. Tim17 is crucial for the switch by performing two separable functions: promotion of inner membrane insertion and binding of Pam18 to form the functional TIM-PAM complex. Thus, the presequence translocase is not a static complex but switches between TOM tethering and PAM binding in a reaction cycle involving Tim21 and Tim17.

Amino Acid Sequence↗

PubMed2005

Pam17 is required for architecture and translocation activity of the mitochondrial protein import motor.

Import of mitochondrial matrix proteins involves the general translocase of the outer membrane and the presequence translocase of the inner membrane. The presequence translocase-associated motor (PAM) drives the completion of preprotein translocation into the matrix. Five subunits of PAM are known: the preprotein-binding matrix heat shock protein 70 (mtHsp70), the nucleotide exchange factor Mge1, Tim44 that directs mtHsp70 to the inner membrane, and the membrane-bound complex of Pam16-Pam18 that regulates the ATPase activity of mtHsp70. We have identified a sixth motor subunit. Pam17 (encoded by the open reading frame YKR065c) is anchored in the inner membrane and exposed to the matrix. Mitochondria lacking Pam17 are selectively impaired in the import of matrix proteins and the generation of an import-driving activity of PAM. Pam17 is required for formation of a stable complex between the cochaperones Pam16 and Pam18 and promotes the association of Pam16-Pam18 with the presequence translocase. Our findings suggest that Pam17 is required for the correct organization of the Pam16-Pam18 complex and thus contributes to regulation of mtHsp70 activity at the inner membrane translocation site.

Amino Acid Sequence↗

PubMed2004

Essential role of Mia40 in import and assembly of mitochondrial intermembrane space proteins.

Mitochondria import nuclear-encoded precursor proteins to four different subcompartments. Specific import machineries have been identified that direct the precursor proteins to the mitochondrial outer membrane, inner membrane or matrix, respectively. However, a machinery dedicated to the import of mitochondrial intermembrane space (IMS) proteins has not been found so far. We have identified the essential IMS protein Mia40 (encoded by the Saccharomyces cerevisiae open reading frame YKL195w). Mitochondria with a mutant form of Mia40 are selectively inhibited in the import of several small IMS proteins, including the essential proteins Tim9 and Tim10. The import of proteins to the other mitochondrial subcompartments does not depend on functional Mia40. The binding of small Tim proteins to Mia40 is crucial for their transport across the outer membrane and represents an initial step in their assembly into IMS complexes. We conclude that Mia40 is a central component of the protein import and assembly machinery of the mitochondrial IMS.

Amino Acid Sequence↗

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

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

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

Machinery for protein sorting and assembly in the mitochondrial outer membrane.

Mitochondria contain translocases for the transport of precursor proteins across their outer and inner membranes. It has been assumed that the translocases also mediate the sorting of proteins to their submitochondrial destination. Here we show that the mitochondrial outer membrane contains a separate sorting and assembly machinery (SAM) that operates after the translocase of the outer membrane (TOM). Mas37 forms a constituent of the SAM complex. The central role of the SAM complex in the sorting and assembly pathway of outer membrane proteins explains the various pleiotropic functions that have been ascribed to Mas37 (refs 4, 11-15). These results suggest that the TOM complex, which can transport all kinds of mitochondrial precursor proteins, is not sufficient for the correct integration of outer membrane proteins with a complicated topology, and instead transfers precursor proteins to the SAM complex.

Gene Deletion↗

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 mitochondrial presequence translocase: an essential role of Tim50 in directing preproteins to the import channel.

Mitochondrial proteins with N-terminal targeting signals are transported across the inner membrane via the presequence translocase, which consists of membrane-integrated channel proteins and the matrix Hsp70 import motor. It has not been known how preproteins are directed to the import channel. We have identified the essential protein Tim50, which exposes its major domain to the intermembrane space. Tim50 interacts with preproteins in transit and directs them to the channel protein Tim23. Inactivation of Tim50 strongly inhibits the import of preproteins with a classical matrix-targeting signal, while preproteins carrying an additional inner membrane-sorting signal do not strictly depend on Tim50. Thus, Tim50 is crucial for guiding the precursors of matrix proteins to their insertion site in the inner membrane.

Amino Acid Sequence↗

PubMed2002

The mitochondrial import machinery: preprotein-conducting channels with binding sites for presequences.

Mitochondrial preproteins with amino-terminal presequences must cross two membranes to reach the matrix of the organelle. Both outer and inner membranes contain hydrophilic high-conductance channels that are responsible for selective translocation of preproteins. The channels are embedded in dynamic protein complexes, the TOM complex of the outer membrane and the TIM23 complex of the inner membrane. Both channel-forming proteins, Tom40 and Tim23, carry specific binding sites for presequences, but differ in their pore size and response to a membrane potential. Studies with the TOM machinery show that other subunits of the translocase complex also provide specific binding sites for preproteins, modulate the channel activity and are critical for assembly of the channel.

Binding Sites↗

PubMed2002

How mitochondria import hydrophilic and hydrophobic proteins.

Most mitochondrial proteins are nuclear encoded and have to be transported into the organelle after synthesis on cytosolic ribosomes. Three multimeric protein complexes have been identified that import precursor proteins destined for the mitochondria: the TOM complex in the outer membrane and two TIM complexes in the inner membrane. Recent work has provided a detailed view of the different mechanisms operating during the import of the two major classes of mitochondrial proteins--hydrophilic proteins with cleavable presequences and hydrophobic proteins with multiple internal signals.

Animals↗