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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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Sensitivity of Tic20 and Tic22 to extraction of envelope  membranes with alkaline buffers. Total envelope membranes  (Env) (50 μg of protein) were incubated in the presence (+pH  11.5) or absence (−pH 11.5) of 0.1 M Na2CO3, pH 11.5, on ice for  10 min. The insoluble (P) and soluble (S) fractions were separated by differential centrifugation and analyzed by (A) fluorography or (B) immunoblotting with anti-Tic22, anti-Tic20, or anti-Toc75 sera as indicated.
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Figure 4: Sensitivity of Tic20 and Tic22 to extraction of envelope membranes with alkaline buffers. Total envelope membranes (Env) (50 μg of protein) were incubated in the presence (+pH 11.5) or absence (−pH 11.5) of 0.1 M Na2CO3, pH 11.5, on ice for 10 min. The insoluble (P) and soluble (S) fractions were separated by differential centrifugation and analyzed by (A) fluorography or (B) immunoblotting with anti-Tic22, anti-Tic20, or anti-Toc75 sera as indicated.

Mentions: To investigate the association of Tic20 and Tic22 with the inner membrane, we tested the sensitivity of both proteins to extraction with alkaline buffer by treating envelope membrane fractions with 0.1 M Na2CO3, pH 11.5. Envelope membranes from a pS-protA cross-linking reaction were used in the extractions to follow both cross-link– labeled (Fig. 4 A) and total immunoreactive (Fig. 4 B) Tic20 and Tic22. Before alkaline extraction, the envelope membranes were treated with dithiothreitol to cleave cross- links that could affect their membrane association. As a control for extraction, Toc75, a known integral envelope membrane protein (Schnell et al., 1994) also was assayed. As expected, Toc75 remains associated with the envelope membranes after alkali extraction (Fig. 4 B). Likewise, Tic20 is completely resistant to extraction from the envelope fraction (Fig. 4, A and B, compare lanes 2 with 3) consistent with the characteristics of an integral membrane protein. In contrast, 40–50% of cross-link–labeled Tic22 is extractable from envelope membranes with alkaline carbonate (Fig. 4, A and B, compare lane 1 with lanes 2 and 3). Based on this analysis, Tic22 appears to be a peripherally associated component of the inner membrane.


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)

Sensitivity of Tic20 and Tic22 to extraction of envelope  membranes with alkaline buffers. Total envelope membranes  (Env) (50 μg of protein) were incubated in the presence (+pH  11.5) or absence (−pH 11.5) of 0.1 M Na2CO3, pH 11.5, on ice for  10 min. The insoluble (P) and soluble (S) fractions were separated by differential centrifugation and analyzed by (A) fluorography or (B) immunoblotting with anti-Tic22, anti-Tic20, or anti-Toc75 sera as indicated.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132967&req=5

Figure 4: Sensitivity of Tic20 and Tic22 to extraction of envelope membranes with alkaline buffers. Total envelope membranes (Env) (50 μg of protein) were incubated in the presence (+pH 11.5) or absence (−pH 11.5) of 0.1 M Na2CO3, pH 11.5, on ice for 10 min. The insoluble (P) and soluble (S) fractions were separated by differential centrifugation and analyzed by (A) fluorography or (B) immunoblotting with anti-Tic22, anti-Tic20, or anti-Toc75 sera as indicated.
Mentions: To investigate the association of Tic20 and Tic22 with the inner membrane, we tested the sensitivity of both proteins to extraction with alkaline buffer by treating envelope membrane fractions with 0.1 M Na2CO3, pH 11.5. Envelope membranes from a pS-protA cross-linking reaction were used in the extractions to follow both cross-link– labeled (Fig. 4 A) and total immunoreactive (Fig. 4 B) Tic20 and Tic22. Before alkaline extraction, the envelope membranes were treated with dithiothreitol to cleave cross- links that could affect their membrane association. As a control for extraction, Toc75, a known integral envelope membrane protein (Schnell et al., 1994) also was assayed. As expected, Toc75 remains associated with the envelope membranes after alkali extraction (Fig. 4 B). Likewise, Tic20 is completely resistant to extraction from the envelope fraction (Fig. 4, A and B, compare lanes 2 with 3) consistent with the characteristics of an integral membrane protein. In contrast, 40–50% of cross-link–labeled Tic22 is extractable from envelope membranes with alkaline carbonate (Fig. 4, A and B, compare lane 1 with lanes 2 and 3). Based on this analysis, Tic22 appears to be a peripherally associated component of the inner membrane.

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