The Structure of Precursor Proteins during Import into Mitochondria

Schwartz, Michael P.; Huang, Shihai; Matouschek, Andreas T

doi:10.1074/jbc.274.18.12759

Cited by 50 publications

(54 citation statements)

References 31 publications

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“…In addition to fusion proteins with these three single disulfide bridges, we constructed all combinations of double disulfide bridge mutants and a fusion protein with all three disulfide bridges present simultaneously. After synthesis of radioactively labeled fusion proteins by in vitro transcription and translation, disulfide bridge formation was induced by oxidation with ferricyanide, as described previously (36). Disulfide bridge formation was complete, and no unreacted cysteine residues could be detected with the sulfhydryl-reactive reagent SDSM (data not shown).…”

Section: Resultsmentioning

confidence: 99%

“…1). To prevent specific parts of the substrate protein from unfolding, we introduced covalent cross-links into the barnase domain by mutating pairs of residues to cysteine at positions that allow disulfide bridge formation upon oxidation (36,37). The disulfide bridges were introduced at three different positions, Cys 43 -Cys 80 , Cys 70 -Cys 92 , and Cys 85 -Cys 102 (the numbering refers to the residue position within the barnase domain).…”

Section: Resultsmentioning

confidence: 99%

“…Because disulfide bonds are covalent cross-links, they persist after a protein is unfolded and introduce a permanent polypeptide loop into the primary structure (see below). Radiolabeled fusion proteins were synthesized by coupled in vitro transcription and translation in E. coli S30 extract, and disulfide bridge formation was induced by oxidation (36). After partial purification, the proteins were incubated in reticulocyte lysate, and their degradation was monitored by SDS-PAGE and electronic autoradiography (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…ClpXP degrades dimeric P22 Arc repressor bearing a C-terminal SsrA tag with similar efficiencies whether or not the two subunits are cross-linked with a disulfide bridge (43). An example of an unrelated protein translocation pore that allows concomitant passage of five polypeptide strands is the mitochondrial protein import channel (36,44).…”

Section: Discussionmentioning

confidence: 99%

“…Under these conditions disulfide bridge formation is complete, and no unreacted cysteine residues could be detected by modification with the sulfhydryl-reactive reagent 4-acetamido-4Ј-maleimidylstilbene-2,2Ј-disulfonic acid (SDSM) followed by SDS-PAGE (36) (data not shown). The proteins were partially purified by high speed centrifugation before cross-linking and by ammonium sulfate precipitation after cross-linking as described previously (36).…”

Section: Methodsmentioning

confidence: 99%

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Concurrent Translocation of Multiple Polypeptide Chains through the Proteasomal Degradation Channel

Lee

Prakash

Matouschek

2002

Journal of Biological Chemistry

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The proteasome can actively unfold proteins by sequentially unraveling their substrates from the attachment point of the degradation signal. To investigate the steric constraints imposed on substrate proteins during their degradation by the proteasome, we constructed a model protein in which specific parts of the polypeptide chain were covalently connected through disulfide bridges. The cross-linked model proteins were fully degraded by the proteasome, but two or more cross-links retarded the degradation slightly. These results suggest that the pore of the proteasome allows the concurrent passage of at least three stretches of a polypeptide chain. A degradation channel that can tolerate some steric bulk may reconcile the two opposing needs for degradation that is compartmentalized to avoid aberrant proteolysis yet able to handle a range of substrates of various sizes.Protein degradation is a critical part of cellular regulation (1). In eukaryotic cells, a multicomponent protease called the proteasome is responsible for the turnover of short-lived regulatory proteins, the removal of abnormal polypeptides, and the production of peptides for antigen presentation (2). Degradation of short-lived regulatory proteins is essential for a wide range of cellular functions including cell cycle control and signal transduction (3). Failure to degrade aggregates of misfolded proteins can lead to disease such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and Huntington's disease (4). Degradation by the proteasome usually involves two consecutive steps: targeting of the substrate for degradation by the attachment of polyubiquitin chains and degradation of the tagged protein by the proteasome with concomitant release of ubiquitin (5).The active sites of proteolysis of the proteasome by themselves show little substrate specificity. Specificity of degradation is achieved by sequestering the proteolytic sites within the structure and tightly controlling access. The three-dimensional structure of the proteasome has been determined by x-ray crystallography and electron microscopy (6 -11). The proteasome consists of a central proteolytic core particle with regulatory caps at either end of it. The core particle is made of two copies of seven different ␣-subunits and seven different ␤-subunits. The subunits are arranged in four heptameric rings, which are stacked on top of each other to form a cylindrical particle. The rings form a central ␤-chamber and two ␣-chambers, one at each end of the particle (2). The proteolytic sites are located in the ␤-chamber and are accessible only through a central channel that runs along the long axis of the particle. The channel has narrow constrictions at the entrance and exit of the ␣-chamber (2). In the isolated yeast core particle, the entrance to the degradation channel is blocked by the N termini of the ␣-subunits (7). The channel opens when the regulatory caps bind to the core (12). The caps consist of 17 subunits, six of which have ATPase activity, and contact the co...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 3 more Smart Citations

Concurrent Translocation of Multiple Polypeptide Chains through the Proteasomal Degradation Channel

Lee

Prakash

Matouschek

2002

Journal of Biological Chemistry

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Proteins Import into Mitochondria

Neupert

Brunner

Hell

2008

Protein Science Encyclopedia

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Originally published in: Protein Folding Handbook. Part II. Edited by Johannes Buchner and Thomas Kiefhaber. Copyright © 2005 Wiley‐VCH Verlag GmbH & Co. KGaA Weinheim. Print ISBN: 3‐527‐30784‐2 The sections in this article are Introduction Translocation Machineries and Pathways of the Mitochondrial Protein Import System Import of Proteins Destined for the Mitochondrial Matrix Import into Mitochondria Requires Protein Unfolding Mechanisms of Unfolding by the Mitochondrial Import Motor Targeted Brownian Ratchet Power‐stroke Model Studies to Discriminate between the Models Studies on the Unfolding of Preproteins Comparison of the Import of Folded and Unfolded Proteins Import of Preproteins With Different Presequence Lengths Import of Titin Domains Unfolding by the Mitochondrial Membrane Potential ΔΨ Mechanistic Studies of the Import Motor Brownian Movement of the Polypeptide Within the Import Channel Recruitment of mtHsp70 by Tim44 Import Without Recruitment of mtHsp70 by Tim44 MtHsp70 Function in the Import Motor Conclusions

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