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Tic20 and Tic22 are new components of the protein import apparatus at the chloroplast inner envelope membrane.

Kouranov A, Chen X, Fuks B, Schnell DJ - J. Cell Biol. (1998)

Bottom Line: In contrast, Tic22 is a 22-kD protein that is located in the intermembrane space between the outer and inner envelope membranes and is peripherally associated with the outer face of the inner membrane.Preprotein import intermediates quantitatively associate with this outer/inner membrane supercomplex, providing evidence that the complex corresponds to envelope contact sites that mediate direct transport of preproteins from the cytoplasm to the stromal compartment.On the basis of these results, we propose that Tic20 and Tic22 are core components of the protein translocon of the inner envelope membrane of chloroplasts.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Rutgers, The State University of New Jersey, Newark, New Jersey 07102, USA.

ABSTRACT
Two components of the chloroplast envelope, Tic20 and Tic22, were previously identified as candidates for components of the general protein import machinery by their ability to covalently cross-link to nuclear-encoded preproteins trapped at an intermediate stage in import across the envelope (Kouranov, A., and D.J. Schnell. 1997. J. Cell Biol. 139:1677-1685). We have determined the primary structures of Tic20 and Tic22 and investigated their localization and association within the chloroplast envelope. Tic20 is a 20-kD integral membrane component of the inner envelope membrane. In contrast, Tic22 is a 22-kD protein that is located in the intermembrane space between the outer and inner envelope membranes and is peripherally associated with the outer face of the inner membrane. Tic20, Tic22, and a third inner membrane import component, Tic110, associate with import components of the outer envelope membrane. Preprotein import intermediates quantitatively associate with this outer/inner membrane supercomplex, providing evidence that the complex corresponds to envelope contact sites that mediate direct transport of preproteins from the cytoplasm to the stromal compartment. On the basis of these results, we propose that Tic20 and Tic22 are core components of the protein translocon of the inner envelope membrane of chloroplasts.

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Resolution of Tic20 and Tic22 by reverse phase chromatography of pS-protA cross-linked envelope membranes.  Chloroplast envelope membranes (300 μg of protein) from pS-protA cross-linked chloroplasts were dissolved in 90% formic  acid and separated by reverse phase chromatography using a  polystyrene matrix and a solvent gradient of 0 to 100% acetonitrile in 0.1% trifluoroacetic acid. Chromatography fractions were  resolved by SDS-PAGE and transfered to nitrocellulose membrane. (A) Imido black stain of reverse phase profile of envelope  membranes. The positions of Tic22, Tic20, and IEP21 are indicated by the horizontal arrow, vertical arrows, and asterisk, respectively. Left, positions of molecular weight standards. (B) Fluorograph of the reverse phase profile shown in A. (C)  Immunoblot of the reverse phase profile shown in A with antisera to Toc75, Toc86, Tic22, and Tic20. The positions of the Toc  and Tic components and pS-protA are shown at the right of B  and C.
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Figure 1: Resolution of Tic20 and Tic22 by reverse phase chromatography of pS-protA cross-linked envelope membranes. Chloroplast envelope membranes (300 μg of protein) from pS-protA cross-linked chloroplasts were dissolved in 90% formic acid and separated by reverse phase chromatography using a polystyrene matrix and a solvent gradient of 0 to 100% acetonitrile in 0.1% trifluoroacetic acid. Chromatography fractions were resolved by SDS-PAGE and transfered to nitrocellulose membrane. (A) Imido black stain of reverse phase profile of envelope membranes. The positions of Tic22, Tic20, and IEP21 are indicated by the horizontal arrow, vertical arrows, and asterisk, respectively. Left, positions of molecular weight standards. (B) Fluorograph of the reverse phase profile shown in A. (C) Immunoblot of the reverse phase profile shown in A with antisera to Toc75, Toc86, Tic22, and Tic20. The positions of the Toc and Tic components and pS-protA are shown at the right of B and C.

Mentions: Antibodies to Toc34, Toc75, Toc86, the small and large subunits of rubisco, and the phosphate-triose phosphate translocator were generated as previously described (Ma et al., 1996; Schnell et al., 1990). Tic110 and cpn60 antibodies were a generous gift of F. Kessler and G. Blobel (both from Rockefeller University, New York, NY). Tic55 antibodies were a generous gift of the laboratory of J. Soll (Christian-Albrechts University, Kiel, Germany). Anti-ClpC serum was a generous gift of the laboratory of K. Keegstra (Michigan State University, East Lansing, MI). The anti-IEP21 serum was prepared to a major 21-kD integral inner envelope membrane protein that had been resolved from the bulk of envelope proteins by sequential reverse phase FPLC and one-dimensional SDS-PAGE. The position of IEP21 is indicated with an asterisk in Fig. 1 A. The anti-Tic20 serum was generated to a 13-amino acid synthetic peptide corresponding to residues 84–96 of the deduced sequence of Toc20 (see Fig. 6 B). The anti-Tic22 serum was generated to full-length recombinant Tic22 that was expressed in Escherichia coli BL21 (DE3) as a fusion to a hexahistidine tag using the pET21d vector system (Novagen, Madison, WI). The recombinant Tic22 was purified on a nickel-chelate matrix according to the supplier's recommendations (Novagen) and was used directly for immunization of rabbits. Immunoblotting with all sera was performed as previously described (Ma et al., 1996) using chemiluminescence detection.


Tic20 and Tic22 are new components of the protein import apparatus at the chloroplast inner envelope membrane.

Kouranov A, Chen X, Fuks B, Schnell DJ - J. Cell Biol. (1998)

Resolution of Tic20 and Tic22 by reverse phase chromatography of pS-protA cross-linked envelope membranes.  Chloroplast envelope membranes (300 μg of protein) from pS-protA cross-linked chloroplasts were dissolved in 90% formic  acid and separated by reverse phase chromatography using a  polystyrene matrix and a solvent gradient of 0 to 100% acetonitrile in 0.1% trifluoroacetic acid. Chromatography fractions were  resolved by SDS-PAGE and transfered to nitrocellulose membrane. (A) Imido black stain of reverse phase profile of envelope  membranes. The positions of Tic22, Tic20, and IEP21 are indicated by the horizontal arrow, vertical arrows, and asterisk, respectively. Left, positions of molecular weight standards. (B) Fluorograph of the reverse phase profile shown in A. (C)  Immunoblot of the reverse phase profile shown in A with antisera to Toc75, Toc86, Tic22, and Tic20. The positions of the Toc  and Tic components and pS-protA are shown at the right of B  and C.
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Figure 1: Resolution of Tic20 and Tic22 by reverse phase chromatography of pS-protA cross-linked envelope membranes. Chloroplast envelope membranes (300 μg of protein) from pS-protA cross-linked chloroplasts were dissolved in 90% formic acid and separated by reverse phase chromatography using a polystyrene matrix and a solvent gradient of 0 to 100% acetonitrile in 0.1% trifluoroacetic acid. Chromatography fractions were resolved by SDS-PAGE and transfered to nitrocellulose membrane. (A) Imido black stain of reverse phase profile of envelope membranes. The positions of Tic22, Tic20, and IEP21 are indicated by the horizontal arrow, vertical arrows, and asterisk, respectively. Left, positions of molecular weight standards. (B) Fluorograph of the reverse phase profile shown in A. (C) Immunoblot of the reverse phase profile shown in A with antisera to Toc75, Toc86, Tic22, and Tic20. The positions of the Toc and Tic components and pS-protA are shown at the right of B and C.
Mentions: Antibodies to Toc34, Toc75, Toc86, the small and large subunits of rubisco, and the phosphate-triose phosphate translocator were generated as previously described (Ma et al., 1996; Schnell et al., 1990). Tic110 and cpn60 antibodies were a generous gift of F. Kessler and G. Blobel (both from Rockefeller University, New York, NY). Tic55 antibodies were a generous gift of the laboratory of J. Soll (Christian-Albrechts University, Kiel, Germany). Anti-ClpC serum was a generous gift of the laboratory of K. Keegstra (Michigan State University, East Lansing, MI). The anti-IEP21 serum was prepared to a major 21-kD integral inner envelope membrane protein that had been resolved from the bulk of envelope proteins by sequential reverse phase FPLC and one-dimensional SDS-PAGE. The position of IEP21 is indicated with an asterisk in Fig. 1 A. The anti-Tic20 serum was generated to a 13-amino acid synthetic peptide corresponding to residues 84–96 of the deduced sequence of Toc20 (see Fig. 6 B). The anti-Tic22 serum was generated to full-length recombinant Tic22 that was expressed in Escherichia coli BL21 (DE3) as a fusion to a hexahistidine tag using the pET21d vector system (Novagen, Madison, WI). The recombinant Tic22 was purified on a nickel-chelate matrix according to the supplier's recommendations (Novagen) and was used directly for immunization of rabbits. Immunoblotting with all sera was performed as previously described (Ma et al., 1996) using chemiluminescence detection.

Bottom Line: In contrast, Tic22 is a 22-kD protein that is located in the intermembrane space between the outer and inner envelope membranes and is peripherally associated with the outer face of the inner membrane.Preprotein import intermediates quantitatively associate with this outer/inner membrane supercomplex, providing evidence that the complex corresponds to envelope contact sites that mediate direct transport of preproteins from the cytoplasm to the stromal compartment.On the basis of these results, we propose that Tic20 and Tic22 are core components of the protein translocon of the inner envelope membrane of chloroplasts.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Rutgers, The State University of New Jersey, Newark, New Jersey 07102, USA.

ABSTRACT
Two components of the chloroplast envelope, Tic20 and Tic22, were previously identified as candidates for components of the general protein import machinery by their ability to covalently cross-link to nuclear-encoded preproteins trapped at an intermediate stage in import across the envelope (Kouranov, A., and D.J. Schnell. 1997. J. Cell Biol. 139:1677-1685). We have determined the primary structures of Tic20 and Tic22 and investigated their localization and association within the chloroplast envelope. Tic20 is a 20-kD integral membrane component of the inner envelope membrane. In contrast, Tic22 is a 22-kD protein that is located in the intermembrane space between the outer and inner envelope membranes and is peripherally associated with the outer face of the inner membrane. Tic20, Tic22, and a third inner membrane import component, Tic110, associate with import components of the outer envelope membrane. Preprotein import intermediates quantitatively associate with this outer/inner membrane supercomplex, providing evidence that the complex corresponds to envelope contact sites that mediate direct transport of preproteins from the cytoplasm to the stromal compartment. On the basis of these results, we propose that Tic20 and Tic22 are core components of the protein translocon of the inner envelope membrane of chloroplasts.

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