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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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Association of Tic20 and Tic22 in the  inner envelope membrane. Isolated mixed outer  and inner envelope membrane vesicles (Env)  were dissolved in TES buffer containing 1% (wt/ vol) Triton X-100. (A) A portion of the soluble  detergent extract (80 μg of protein) was subjected to immunoaffinity chromatography on  anti-Tic22 Sepharose (anti-Tic22) or preimmune  IgG Sepharose (PI). The unbound (U) and  bound (B) fractions from the chromatography  runs were analyzed by SDS-PAGE and immunoblotting with antisera to chloroplast proteins as  indicated. Lane 1 contains one-fourth of the total  envelope membrane protein (20 μg) applied to  the Sepharose columns. (B) The soluble detergent extract (200 μg of protein) was resolved by  FPLC on a Superose 6HR column (Pharmacia)  equilibrated in a buffer containing 0.1% Triton  X-100. Fractions of 1 ml in volume were collected  and analyzed by SDS-PAGE and immunoblotting with antisera against Tic20, Tic22, cpn60, triose phosphate–phosphate translocator (PT), a  21-kD inner envelope membrane protein  (IEP21), and the small subunit of rubisco  (rubisco).
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Figure 7: Association of Tic20 and Tic22 in the inner envelope membrane. Isolated mixed outer and inner envelope membrane vesicles (Env) were dissolved in TES buffer containing 1% (wt/ vol) Triton X-100. (A) A portion of the soluble detergent extract (80 μg of protein) was subjected to immunoaffinity chromatography on anti-Tic22 Sepharose (anti-Tic22) or preimmune IgG Sepharose (PI). The unbound (U) and bound (B) fractions from the chromatography runs were analyzed by SDS-PAGE and immunoblotting with antisera to chloroplast proteins as indicated. Lane 1 contains one-fourth of the total envelope membrane protein (20 μg) applied to the Sepharose columns. (B) The soluble detergent extract (200 μg of protein) was resolved by FPLC on a Superose 6HR column (Pharmacia) equilibrated in a buffer containing 0.1% Triton X-100. Fractions of 1 ml in volume were collected and analyzed by SDS-PAGE and immunoblotting with antisera against Tic20, Tic22, cpn60, triose phosphate–phosphate translocator (PT), a 21-kD inner envelope membrane protein (IEP21), and the small subunit of rubisco (rubisco).

Mentions: The ability of Tic20 and Tic22 to cross-link to translocating polypeptides (Ma et al., 1996; Kouranov and Schnell, 1997) raises the possibility that they may form part of a stable membrane complex at the core of a translocon in the inner membrane. As a first step to explore the nature of the inner membrane translocon, we investigated the interactions of Tic22 and Tic20 with each other. Total chloroplast envelope membranes were dissolved under mild conditions with Triton X-100 to maintain the native structure of import complexes and proteins were immunoaffinity purified with anti-Tic22 IgG Sepharose or preimmune IgG Sepharose. The IgG-bound fractions were resolved by SDS-PAGE and immunoblotted with anti-Tic20 and anti- Tic22. As expected, Tic22 was bound by anti-Tic22, but not by IgG from its corresponding preimmune sera (Fig. 7 A, compare lanes 3 with 4). Tic20 also is detected in the anti-Tic22–bound fraction (Fig. 7 A, lane 3), suggesting that these two components form a stable association in the inner membrane. As controls for the specificity of the immunoaffinity reaction, the anti-Tic22 Sepharose chromatography fractions were immunoblotted with antisera to the triose phosphate–phosphate translocator, an inner membrane metabolite transporter (Fliege et al., 1978), and IEP21, an integral inner membrane protein (Materials and Methods). In addition, the immunoblot was probed with antiserum to the large subunit of rubisco, a common stromal contaminant of the envelope membranes. None of the three control proteins were detected in the bound fraction confirming the selectivity of the anti-Tic22 Sepharose (Fig. 7 A).


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)

Association of Tic20 and Tic22 in the  inner envelope membrane. Isolated mixed outer  and inner envelope membrane vesicles (Env)  were dissolved in TES buffer containing 1% (wt/ vol) Triton X-100. (A) A portion of the soluble  detergent extract (80 μg of protein) was subjected to immunoaffinity chromatography on  anti-Tic22 Sepharose (anti-Tic22) or preimmune  IgG Sepharose (PI). The unbound (U) and  bound (B) fractions from the chromatography  runs were analyzed by SDS-PAGE and immunoblotting with antisera to chloroplast proteins as  indicated. Lane 1 contains one-fourth of the total  envelope membrane protein (20 μg) applied to  the Sepharose columns. (B) The soluble detergent extract (200 μg of protein) was resolved by  FPLC on a Superose 6HR column (Pharmacia)  equilibrated in a buffer containing 0.1% Triton  X-100. Fractions of 1 ml in volume were collected  and analyzed by SDS-PAGE and immunoblotting with antisera against Tic20, Tic22, cpn60, triose phosphate–phosphate translocator (PT), a  21-kD inner envelope membrane protein  (IEP21), and the small subunit of rubisco  (rubisco).
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Figure 7: Association of Tic20 and Tic22 in the inner envelope membrane. Isolated mixed outer and inner envelope membrane vesicles (Env) were dissolved in TES buffer containing 1% (wt/ vol) Triton X-100. (A) A portion of the soluble detergent extract (80 μg of protein) was subjected to immunoaffinity chromatography on anti-Tic22 Sepharose (anti-Tic22) or preimmune IgG Sepharose (PI). The unbound (U) and bound (B) fractions from the chromatography runs were analyzed by SDS-PAGE and immunoblotting with antisera to chloroplast proteins as indicated. Lane 1 contains one-fourth of the total envelope membrane protein (20 μg) applied to the Sepharose columns. (B) The soluble detergent extract (200 μg of protein) was resolved by FPLC on a Superose 6HR column (Pharmacia) equilibrated in a buffer containing 0.1% Triton X-100. Fractions of 1 ml in volume were collected and analyzed by SDS-PAGE and immunoblotting with antisera against Tic20, Tic22, cpn60, triose phosphate–phosphate translocator (PT), a 21-kD inner envelope membrane protein (IEP21), and the small subunit of rubisco (rubisco).
Mentions: The ability of Tic20 and Tic22 to cross-link to translocating polypeptides (Ma et al., 1996; Kouranov and Schnell, 1997) raises the possibility that they may form part of a stable membrane complex at the core of a translocon in the inner membrane. As a first step to explore the nature of the inner membrane translocon, we investigated the interactions of Tic22 and Tic20 with each other. Total chloroplast envelope membranes were dissolved under mild conditions with Triton X-100 to maintain the native structure of import complexes and proteins were immunoaffinity purified with anti-Tic22 IgG Sepharose or preimmune IgG Sepharose. The IgG-bound fractions were resolved by SDS-PAGE and immunoblotted with anti-Tic20 and anti- Tic22. As expected, Tic22 was bound by anti-Tic22, but not by IgG from its corresponding preimmune sera (Fig. 7 A, compare lanes 3 with 4). Tic20 also is detected in the anti-Tic22–bound fraction (Fig. 7 A, lane 3), suggesting that these two components form a stable association in the inner membrane. As controls for the specificity of the immunoaffinity reaction, the anti-Tic22 Sepharose chromatography fractions were immunoblotted with antisera to the triose phosphate–phosphate translocator, an inner membrane metabolite transporter (Fliege et al., 1978), and IEP21, an integral inner membrane protein (Materials and Methods). In addition, the immunoblot was probed with antiserum to the large subunit of rubisco, a common stromal contaminant of the envelope membranes. None of the three control proteins were detected in the bound fraction confirming the selectivity of the anti-Tic22 Sepharose (Fig. 7 A).

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.

Show MeSH
Related in: MedlinePlus