Translocation arrest by reversible folding of a precursor protein imported into mitochondria. A means to quantitate translocation contact sites

doi:10.1083/jcb.109.4.1421

This Article

	Full Text (PDF, 1618K)
	Alert me when this article is cited
	Citation Map

Services

	Email this article
	Similar articles in this journal
	Similar articles in PubMed
	Alert me to new content in the JCB
	Download to citation manager

Citing Articles

	Citing Articles via HighWire
	Citing Articles via CrossRef
	Citing Articles via Google Scholar

Google Scholar

	Articles by Rassow, J.
	Articles by Neupert, W.
	Search for Related Content

PubMed

	PubMed Citation
	Articles by Rassow, J.
	Articles by Neupert, W.


Social Bookmarking

	What's this?

ARTICLES

Translocation arrest by reversible folding of a precursor protein imported into mitochondria. A means to quantitate translocation contact sites

J Rassow, B Guiard, U Wienhues, V Herzog, FU Hartl and W Neupert
Institut fur Physiologische Chemie, Zellbiologie und Physikalische Biochemie der Universitat Munchen, Federal Republic of Germany.

Passage of precursor proteins through translocation contact sites of mitochondria was investigated by studying the import of a fusion protein consisting of the NH2-terminal 167 amino acids of yeast cytochrome b2 precursor and the complete mouse dihydrofolate reductase. Isolated mitochondria of Neurospora crassa readily imported the fusion protein. In the presence of methotrexate import was halted and a stable intermediate spanning both mitochondrial membranes at translocation contact sites accumulated. The complete dihydrofolate reductase moiety in this intermediate was external to the outer membrane, and the 136 amino acid residues of the cytochrome b2 moiety remaining after cleavage by the matrix processing peptidase spanned both outer and inner membranes. Removal of methotrexate led to import of the intermediate retained at the contact site into the matrix. Thus unfolding at the surface of the outer mitochondrial membrane is a prerequisite for passage through translocation contact sites. The membrane-spanning intermediate was used to estimate the number of translocation sites. Saturation was reached at 70 pmol intermediate per milligram of mitochondrial protein. This amount of translocation intermediates was calculated to occupy approximately 1% of the total surface of the outer membrane. The morphometrically determined area of close contact between outer and inner membranes corresponded to approximately 7% of the total outer membrane surface. Accumulation of the intermediate inhibited the import of other precursor proteins suggesting that different precursor proteins are using common translocation contact sites. We conclude that the machinery for protein translocation into mitochondria is present at contact sites in limited number.
Add to CiteULike CiteULike Add to Complore Complore Add to Connotea Connotea Add to Del.icio.us Del.icio.us Add to Digg Digg Facebook Add to Reddit Reddit Add to Technorati Technorati Twitter What's this?

This article has been cited by other articles:

Wagner, K., Gebert, N., Guiard, B., Brandner, K., Truscott, K. N., Wiedemann, N., Pfanner, N., Rehling, P. (2008). The Assembly Pathway of the Mitochondrial Carrier Translocase Involves Four Preprotein Translocases. Mol. Cell. Biol. 28: 4251-4260 [Abstract] [Full Text]
Krayl, M., Lim, J. H., Martin, F., Guiard, B., Voos, W. (2007). A Cooperative Action of the ATP-Dependent Import Motor Complex and the Inner Membrane Potential Drives Mitochondrial Preprotein Import. Mol. Cell. Biol. 27: 411-425 [Abstract] [Full Text]
Vogel, F., Bornhovd, C., Neupert, W., Reichert, A. S. (2006). Dynamic subcompartmentalization of the mitochondrial inner membrane. JCB 175: 237-247 [Abstract] [Full Text]
Meinecke, M., Wagner, R., Kovermann, P., Guiard, B., Mick, D. U., Hutu, D. P., Voos, W., Truscott, K. N., Chacinska, A., Pfanner, N., Rehling, P. (2006). Tim50 maintains the permeability barrier of the mitochondrial inner membrane.. Science 312: 1523-1526 [Abstract] [Full Text]
Gu, Y., Gordon, D. M., Amutha, B., Pain, D. (2005). A GTP:AMP Phosphotransferase, Adk2p, in Saccharomyces cerevisiae: ROLE OF THE C TERMINUS IN PROTEIN FOLDING/STABILIZATION, THERMAL TOLERANCE, AND ENZYMATIC ACTIVITY. J. Biol. Chem. 280: 18604-18609 [Abstract] [Full Text]
Heller, U., Winklhofer, K. F., Heske, J., Reintjes, A., Tatzelt, J. (2003). Post-translational Import of the Prion Protein into the Endoplasmic Reticulum Interferes with Cell Viability: A CRITICAL ROLE FOR THE PUTATIVE TRANSMEMBRANE DOMAIN. J. Biol. Chem. 278: 36139-36147 [Abstract] [Full Text]
Moro, F., Okamoto, K., Donzeau, M., Neupert, W., Brunner, M. (2002). Mitochondrial Protein Import: Molecular Basis of the ATP-dependent Interaction of MtHsp70 with Tim44. J. Biol. Chem. 277: 6874-6880 [Abstract] [Full Text]
Tanudji, M., Hevi, S., Chuck, S. L. (2002). Improperly folded green fluorescent protein is secreted via a non-classical pathway. J. Cell Sci. 115: 3849-3857 [Abstract] [Full Text]
Sinai, A. P., Joiner, K. A. (2001). The Toxoplasma gondii protein ROP2 mediates host organelle association with the parasitophorous vacuole membrane. JCB 154: 95-108 [Abstract] [Full Text]
Meisinger, C., Ryan, M. T., Hill, K., Model, K., Lim, J. H., Sickmann, A., Müller, H., Meyer, H. E., Wagner, R., Pfanner, N. (2001). Protein Import Channel of the Outer Mitochondrial Membrane: a Highly Stable Tom40-Tom22 Core Structure Differentially Interacts with Preproteins, Small Tom Proteins, and Import Receptors. Mol. Cell. Biol. 21: 2337-2348 [Abstract] [Full Text]
Fritz, S., Rapaport, D., Klanner, E., Neupert, W., Westermann, B. (2001). Connection of the Mitochondrial Outer and Inner Membranes by Fzo1 Is Critical for Organellar Fusion. JCB 152: 683-692 [Abstract] [Full Text]
Geissler, A., Krimmer, T., Bömer, U., Guiard, B., Rassow, J., Pfanner, N. (2000). Membrane Potential-Driven Protein Import into Mitochondria. The Sorting Sequence of Cytochrome b2 Modulates the Delta psi -Dependence of Translocation of the Matrix-targeting Sequence. Mol. Biol. Cell 11: 3977-3991 [Abstract] [Full Text]
Schulke, N., Sepuri, N. B. V., Gordon, D. M., Saxena, S., Dancis, A., Pain, D. (1999). A Multisubunit Complex of Outer and Inner Mitochondrial Membrane Protein Translocases Stabilized in Vivo by Translocation Intermediates. J. Biol. Chem. 274: 22847-22854 [Abstract] [Full Text]
Ryan, M. T., Muller, H., Pfanner, N. (1999). Functional Staging of ADP/ATP Carrier Translocation across the Outer Mitochondrial Membrane. J. Biol. Chem. 274: 20619-20627 [Abstract] [Full Text]
Schulke, N., Sepuri, N. B. V., Pain, D. (1997). In vivo zippering of inner and outer mitochondrial membranes by a stable translocation�intermediate. Proc. Natl. Acad. Sci. USA 94: 7314-7319 [Abstract] [Full Text]
Froehlich, J. E., Keegstra, K. (1997). Identification of a Translocation Intermediate Occupying Functional Protein Import Sites in the Chloroplastic Envelope Membrane. J. Biol. Chem. 272: 8077-8082 [Abstract] [Full Text]
Kanamori, T., Nishikawa, S.-I., Shin, I., Schultz, P.�G., Endo, T. (1997). Probing the environment along the protein import pathways in yeast mitochondria by site-specific�photocrosslinking. Proc. Natl. Acad. Sci. USA 94: 485-490 [Abstract] [Full Text]
Brunner, M., Schneider, H.-C., Lill, R., Neupert, W. (1995). Dissection of Protein Translocation across the Mitochondrial Outer and Inner Membranes. Cold Spring Harb Symp Quant Biol 60: 619-627 [Abstract]
Tian, F., Ma, Y., Kouranov, A., LaSala, S.E., Schnell, D.J. (1995). Molecular Dissection of the Mechanism of Protein Import into Chloroplasts. Cold Spring Harb Symp Quant Biol 60: 629-636 [Abstract]