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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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Sequential immunoaffinity chromatography  of solubilized chloroplast envelope membranes. Isolated  mixed outer and inner chloroplast envelope membranes  (OM/IM) (100 μg of protein)  from untreated chloroplasts  or chloroplasts that had been  cross-linked to 400 nM pS-1  in the presence of 0.1 mM  ATP and GTP were dissolved in TES buffer containing 1% Triton X-100, clarified by centrifugation, and  sequentially passed over anti-Toc34 Sepharose (α-Toc34),  anti-Toc86 Sepharose  (α-Toc86), and anti-Tic110  Sepharose (α-Tic110). The  eluates and unbound (unbound) fractions were resolved by SDS-PAGE. The  resolved polypeptides were  visualized directly by Coomassie blue staining (A1 and  B1), immunoblotting with antisera to chloroplast proteins  as indicated (A2 and B2), or  analyzed by phosphorimaging (B3). The immunoblots in  A2 and B2 were performed  with Toc or Tic antisera as indicated and with antisera to  ClpC, cpn60, triose phosphate-phosphate translocator (PT), a 21-kD inner envelope membrane protein  (IEP21), and the small subunit of rubisco (rubisco). The  position of the heavy chain of  IgG (IgG HC) that dissociates from IgG Sepharose during chromatography is indicated between A1 and B1.
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Figure 8: Sequential immunoaffinity chromatography of solubilized chloroplast envelope membranes. Isolated mixed outer and inner chloroplast envelope membranes (OM/IM) (100 μg of protein) from untreated chloroplasts or chloroplasts that had been cross-linked to 400 nM pS-1 in the presence of 0.1 mM ATP and GTP were dissolved in TES buffer containing 1% Triton X-100, clarified by centrifugation, and sequentially passed over anti-Toc34 Sepharose (α-Toc34), anti-Toc86 Sepharose (α-Toc86), and anti-Tic110 Sepharose (α-Tic110). The eluates and unbound (unbound) fractions were resolved by SDS-PAGE. The resolved polypeptides were visualized directly by Coomassie blue staining (A1 and B1), immunoblotting with antisera to chloroplast proteins as indicated (A2 and B2), or analyzed by phosphorimaging (B3). The immunoblots in A2 and B2 were performed with Toc or Tic antisera as indicated and with antisera to ClpC, cpn60, triose phosphate-phosphate translocator (PT), a 21-kD inner envelope membrane protein (IEP21), and the small subunit of rubisco (rubisco). The position of the heavy chain of IgG (IgG HC) that dissociates from IgG Sepharose during chromatography is indicated between A1 and B1.

Mentions: The eluates and unbound fraction from the chromatographic runs were resolved by SDS-PAGE and analyzed by Coomassie blue staining and immunoblotting as shown in Fig. 8. As controls for the selectivity of the immunoaffinity chromatography presented, we probed the immunoblots with antisera to the triose phosphate–phosphate translocator, the large or small subunits of rubisco, and IEP21. None of these control proteins were detected in eluates from the three sequential immunoaffinity columns indicating the selectivity of their respective antigens (Fig. 8, A2 and B2). In addition, IgG Sepharose prepared from a mixture of anti-Toc34, anti-Toc86, and anti-Tic110 preimmune sera did not bind any of the chloroplast proteins as assayed by immunoblotting (data not shown).


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)

Sequential immunoaffinity chromatography  of solubilized chloroplast envelope membranes. Isolated  mixed outer and inner chloroplast envelope membranes  (OM/IM) (100 μg of protein)  from untreated chloroplasts  or chloroplasts that had been  cross-linked to 400 nM pS-1  in the presence of 0.1 mM  ATP and GTP were dissolved in TES buffer containing 1% Triton X-100, clarified by centrifugation, and  sequentially passed over anti-Toc34 Sepharose (α-Toc34),  anti-Toc86 Sepharose  (α-Toc86), and anti-Tic110  Sepharose (α-Tic110). The  eluates and unbound (unbound) fractions were resolved by SDS-PAGE. The  resolved polypeptides were  visualized directly by Coomassie blue staining (A1 and  B1), immunoblotting with antisera to chloroplast proteins  as indicated (A2 and B2), or  analyzed by phosphorimaging (B3). The immunoblots in  A2 and B2 were performed  with Toc or Tic antisera as indicated and with antisera to  ClpC, cpn60, triose phosphate-phosphate translocator (PT), a 21-kD inner envelope membrane protein  (IEP21), and the small subunit of rubisco (rubisco). The  position of the heavy chain of  IgG (IgG HC) that dissociates from IgG Sepharose during chromatography is indicated between A1 and B1.
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Figure 8: Sequential immunoaffinity chromatography of solubilized chloroplast envelope membranes. Isolated mixed outer and inner chloroplast envelope membranes (OM/IM) (100 μg of protein) from untreated chloroplasts or chloroplasts that had been cross-linked to 400 nM pS-1 in the presence of 0.1 mM ATP and GTP were dissolved in TES buffer containing 1% Triton X-100, clarified by centrifugation, and sequentially passed over anti-Toc34 Sepharose (α-Toc34), anti-Toc86 Sepharose (α-Toc86), and anti-Tic110 Sepharose (α-Tic110). The eluates and unbound (unbound) fractions were resolved by SDS-PAGE. The resolved polypeptides were visualized directly by Coomassie blue staining (A1 and B1), immunoblotting with antisera to chloroplast proteins as indicated (A2 and B2), or analyzed by phosphorimaging (B3). The immunoblots in A2 and B2 were performed with Toc or Tic antisera as indicated and with antisera to ClpC, cpn60, triose phosphate-phosphate translocator (PT), a 21-kD inner envelope membrane protein (IEP21), and the small subunit of rubisco (rubisco). The position of the heavy chain of IgG (IgG HC) that dissociates from IgG Sepharose during chromatography is indicated between A1 and B1.
Mentions: The eluates and unbound fraction from the chromatographic runs were resolved by SDS-PAGE and analyzed by Coomassie blue staining and immunoblotting as shown in Fig. 8. As controls for the selectivity of the immunoaffinity chromatography presented, we probed the immunoblots with antisera to the triose phosphate–phosphate translocator, the large or small subunits of rubisco, and IEP21. None of these control proteins were detected in eluates from the three sequential immunoaffinity columns indicating the selectivity of their respective antigens (Fig. 8, A2 and B2). In addition, IgG Sepharose prepared from a mixture of anti-Toc34, anti-Toc86, and anti-Tic110 preimmune sera did not bind any of the chloroplast proteins as assayed by immunoblotting (data not shown).

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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