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AIP is a mitochondrial import mediator that binds to both import receptor Tom20 and preproteins

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      Abstract

      Most mitochondrial preproteins are maintained in a loosely folded import-competent conformation by cytosolic chaperones, and are imported into mitochondria by translocator complexes containing a preprotein receptor, termed translocase of the outer membrane of mitochondria (Tom) 20. Using two-hybrid screening, we identified arylhydrocarbon receptor–interacting protein (AIP), an FK506-binding protein homologue, interacting with Tom20. The extreme COOH-terminal acidic segment of Tom20 was required for interaction with tetratricopeptide repeats of AIP. An in vitro import assay indicated that AIP prevents preornithine transcarbamylase from the loss of import competency. In cultured cells, overexpression of AIP enhanced preornithine transcarbamylase import, and depletion of AIP by RNA interference impaired the import. An in vitro binding assay revealed that AIP specifically binds to mitochondrial preproteins. Formation of a ternary complex of Tom20, AIP, and preprotein was observed. Hsc70 was also found to bind to AIP. An aggregation suppression assay indicated that AIP has a chaperone-like activity to prevent substrate proteins from aggregation. These results suggest that AIP functions as a cytosolic factor that mediates preprotein import into mitochondria.

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      Efficient selection for high-expression transfectants with a novel eukaryotic vector.

      We have developed a new expression vector which allows efficient selection for transfectants that express foreign genes at high levels. The vector is composed of a ubiquitously strong promoter based on the beta-actin promoter, a 69% subregion of the bovine papilloma virus genome, and a mutant neomycin phosphotransferase II-encoding gene driven by a weak promoter, which confers only marginal resistance to G418. Thus, high concentrations of G418 (approx. 800 micrograms/ml) effectively select for transfectants containing a high vector copy number (greater than 300). We tested this system by producing human interleukin-2 (IL-2) in L cells and Chinese hamster ovary (CHO) cells, and the results showed that high concentrations of G418 efficiently yielded L cell and CHO cell transfectants stably producing IL-2 at levels comparable with those previously attained using gene amplification. The vector sequences were found to have integrated into the host chromosome, and were stably maintained in the transfectants for several months.
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        Molecular chaperones Hsp90 and Hsp70 deliver preproteins to the mitochondrial import receptor Tom70.

        The role of cytosolic factors in protein targeting to mitochondria is poorly understood. Here, we show that in mammals, the cytosolic chaperones Hsp90 and Hsp70 dock onto a specialized TPR domain in the import receptor Tom70 at the outer mitochondrial membrane. This interaction serves to deliver a set of preproteins to the receptor for subsequent membrane translocation dependent on the Hsp90 ATPase. Disruption of the chaperone/Tom70 recognition inhibits the import of these preproteins into mitochondria. In yeast, Hsp70 rather than Hsp90 is used in import, and Hsp70 docking is required for the formation of a productive preprotein/Tom70 complex. We outline a novel mechanism in which chaperones are recruited for a specific targeting event by a membrane-bound receptor.
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          Protein import into mitochondria.

           W Neupert (1996)
          Mitochondria import many hundreds of different proteins that are encoded by nuclear genes. These proteins are targeted to the mitochondria, translocated through the mitochondrial membranes, and sorted to the different mitochondrial subcompartments. Separate translocases in the mitochondrial outer membrane (TOM complex) and in the inner membrane (TIM complex) facilitate recognition of preproteins and transport across the two membranes. Factors in the cytosol assist in targeting of preproteins. Protein components in the matrix partake in energetically driving translocation in a reaction that depends on the membrane potential and matrix-ATP. Molecular chaperones in the matrix exert multiple functions in translocation, sorting, folding, and assembly of newly imported proteins.
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            Author and article information

            Affiliations
            Department of Molecular Genetics, Graduate School of Medical Sciences, Kumamoto University, Kumamoto 860-8556, Japan
            Author notes

            Address correspondence to Masato Yano, Department of Molecular Genetics, Graduate School of Medical Sciences, Kumamoto University, Honjo 1-1-1, Kumamoto 860-8556, Japan. Tel.: 81-96-373-5140. Fax: 81-96-373-5145. email: myano@123456gpo.kumamoto-u.ac.jp; or Masataka Mori, Department of Molecular Genetics, Graduate School of Medical Sciences, Kumamoto University, Honjo 1-1-1, Kumamoto 860-8556, Japan. Tel.: 81-96-373-5140. Fax: 81-96-373-5145. email: masa@123456gpo.kumamoto-u.ac.jp

            Journal
            J Cell Biol
            The Journal of Cell Biology
            The Rockefeller University Press
            0021-9525
            1540-8140
            13 October 2003
            : 163
            : 1
            : 45-56
            2173431
            200305051
            10.1083/jcb.200305051
            14557246
            Copyright © 2003, The Rockefeller University Press
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