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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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Protease sensitivity of  Tic20 and Tic22 in intact chloroplasts. Chloroplasts equivalent to  2 mg of chlorophyll were treated  with 200 μg/ml thermolysin or  trypsin on ice for 30 min. The chloroplasts were reisolated through  Percoll silica gel, lysed, and then  subfractionated by flotation into sucrose gradients. Fractions corresponding to envelope membranes  were pooled and analyzed by SDS/ PAGE. (A) Immunoblot of SDS-PAGE resolved envelope polypeptides from chloroplasts incubated in  the absence (−T-lysin) or presence  (+T-lysin) of thermolysin or the  absence (−Trypsin) or presence  (+Trypsin) of trypsin with anti-Toc86, anti-Tic110, anti-Tic20, and anti-Tic22 sera. Each lane contains 50 μg of protein. (B) Immunoblot of chloroplast envelopes with  anti-Tic20 or Tic22 serum after treatment of membranes in the absence or presence of trypsin or thermolysin and the absence (−TX-100) or presence (+TX-100) of Triton X-100. Right, positions of the Toc and Tic components.
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Figure 5: Protease sensitivity of Tic20 and Tic22 in intact chloroplasts. Chloroplasts equivalent to 2 mg of chlorophyll were treated with 200 μg/ml thermolysin or trypsin on ice for 30 min. The chloroplasts were reisolated through Percoll silica gel, lysed, and then subfractionated by flotation into sucrose gradients. Fractions corresponding to envelope membranes were pooled and analyzed by SDS/ PAGE. (A) Immunoblot of SDS-PAGE resolved envelope polypeptides from chloroplasts incubated in the absence (−T-lysin) or presence (+T-lysin) of thermolysin or the absence (−Trypsin) or presence (+Trypsin) of trypsin with anti-Toc86, anti-Tic110, anti-Tic20, and anti-Tic22 sera. Each lane contains 50 μg of protein. (B) Immunoblot of chloroplast envelopes with anti-Tic20 or Tic22 serum after treatment of membranes in the absence or presence of trypsin or thermolysin and the absence (−TX-100) or presence (+TX-100) of Triton X-100. Right, positions of the Toc and Tic components.

Mentions: To confirm the reactivity of the anti-Tic22 serum, we tested the ability of the antibodies to recognize the 22-kD cross-linked product. To avoid complications due to the presence of the protein A IgG-binding domains on the pS-protA cross-linking substrate, cross-linking was performed with pS-1 which lacks the IgG binding domain (Ma et al., 1996). The position of cross-linked Tic22 is largely obscured by [125I]pS-1 on SDS-PAGE gels of envelope membranes because of their similar mobility (Fig. 2 A, lane 1). However, cross-linked Tic22 is visible if chloroplasts are treated with exogenous thermolysin after the cross-linking reaction (Fig. 2 A, lane 2). Thermolysin degrades envelope-bound [125I]pS-1, but does not degrade Tic22 (see Fig. 5). Total envelope membranes from a pS-1 cross-linking experiment performed in the presence of 0.1 mM ATP and GTP were treated with reducing agent, dissolved under denaturing conditions, and then applied to anti-Tic22 IgG Sepharose. Fig. 2 B, lane 1 shows that the radioactive 22-kD polypeptide band corresponding to cross-linked–labeled Tic22 bound to the anti-Tic22 IgGs. The corresponding preimmune IgGs of anti-Tic22 did not bind any radioactively labeled proteins (Fig. 2 B, lane 2). These data confirm the reactivity of the antiserum with Tic22.


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)

Protease sensitivity of  Tic20 and Tic22 in intact chloroplasts. Chloroplasts equivalent to  2 mg of chlorophyll were treated  with 200 μg/ml thermolysin or  trypsin on ice for 30 min. The chloroplasts were reisolated through  Percoll silica gel, lysed, and then  subfractionated by flotation into sucrose gradients. Fractions corresponding to envelope membranes  were pooled and analyzed by SDS/ PAGE. (A) Immunoblot of SDS-PAGE resolved envelope polypeptides from chloroplasts incubated in  the absence (−T-lysin) or presence  (+T-lysin) of thermolysin or the  absence (−Trypsin) or presence  (+Trypsin) of trypsin with anti-Toc86, anti-Tic110, anti-Tic20, and anti-Tic22 sera. Each lane contains 50 μg of protein. (B) Immunoblot of chloroplast envelopes with  anti-Tic20 or Tic22 serum after treatment of membranes in the absence or presence of trypsin or thermolysin and the absence (−TX-100) or presence (+TX-100) of Triton X-100. Right, positions of the Toc and Tic components.
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Figure 5: Protease sensitivity of Tic20 and Tic22 in intact chloroplasts. Chloroplasts equivalent to 2 mg of chlorophyll were treated with 200 μg/ml thermolysin or trypsin on ice for 30 min. The chloroplasts were reisolated through Percoll silica gel, lysed, and then subfractionated by flotation into sucrose gradients. Fractions corresponding to envelope membranes were pooled and analyzed by SDS/ PAGE. (A) Immunoblot of SDS-PAGE resolved envelope polypeptides from chloroplasts incubated in the absence (−T-lysin) or presence (+T-lysin) of thermolysin or the absence (−Trypsin) or presence (+Trypsin) of trypsin with anti-Toc86, anti-Tic110, anti-Tic20, and anti-Tic22 sera. Each lane contains 50 μg of protein. (B) Immunoblot of chloroplast envelopes with anti-Tic20 or Tic22 serum after treatment of membranes in the absence or presence of trypsin or thermolysin and the absence (−TX-100) or presence (+TX-100) of Triton X-100. Right, positions of the Toc and Tic components.
Mentions: To confirm the reactivity of the anti-Tic22 serum, we tested the ability of the antibodies to recognize the 22-kD cross-linked product. To avoid complications due to the presence of the protein A IgG-binding domains on the pS-protA cross-linking substrate, cross-linking was performed with pS-1 which lacks the IgG binding domain (Ma et al., 1996). The position of cross-linked Tic22 is largely obscured by [125I]pS-1 on SDS-PAGE gels of envelope membranes because of their similar mobility (Fig. 2 A, lane 1). However, cross-linked Tic22 is visible if chloroplasts are treated with exogenous thermolysin after the cross-linking reaction (Fig. 2 A, lane 2). Thermolysin degrades envelope-bound [125I]pS-1, but does not degrade Tic22 (see Fig. 5). Total envelope membranes from a pS-1 cross-linking experiment performed in the presence of 0.1 mM ATP and GTP were treated with reducing agent, dissolved under denaturing conditions, and then applied to anti-Tic22 IgG Sepharose. Fig. 2 B, lane 1 shows that the radioactive 22-kD polypeptide band corresponding to cross-linked–labeled Tic22 bound to the anti-Tic22 IgGs. The corresponding preimmune IgGs of anti-Tic22 did not bind any radioactively labeled proteins (Fig. 2 B, lane 2). These data confirm the reactivity of the antiserum with Tic22.

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