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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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Immunoaffinity purification of [125I]Tic22 with anti-Tic22 IgG Sepharose. Intact chloroplasts were cross-linked to  pS-1 in the presence of 0.1 mM ATP and GTP. After import, the  chloroplasts were treated in the absence or presence of 100 μg/ml  thermolysin for 30 min on ice to remove bound pS-1, and chloroplast were subfractionated to yield a total envelope membrane  fraction. Envelope membranes (80 μg of protein) from cross-linked chloroplasts were dissolved under denaturing conditions,  and applied to anti-Tic22 Sepharose (α-Tic22) or preimmune IgG  Sepharose (PI) as indicated. (A) Fluorograph of SDS-PAGE resolved thermolysin-treated or -untreated envelope membranes  (50 μg of protein). The position of Tic and Toc components and  pS-1 are indicated to the left of the figure. (B) Fluorograph of  SDS-PAGE resolved immunoaffinity eluates of anti-Tic22 IgG  Sepharose and preimmune IgG Sepharose.
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Figure 2: Immunoaffinity purification of [125I]Tic22 with anti-Tic22 IgG Sepharose. Intact chloroplasts were cross-linked to pS-1 in the presence of 0.1 mM ATP and GTP. After import, the chloroplasts were treated in the absence or presence of 100 μg/ml thermolysin for 30 min on ice to remove bound pS-1, and chloroplast were subfractionated to yield a total envelope membrane fraction. Envelope membranes (80 μg of protein) from cross-linked chloroplasts were dissolved under denaturing conditions, and applied to anti-Tic22 Sepharose (α-Tic22) or preimmune IgG Sepharose (PI) as indicated. (A) Fluorograph of SDS-PAGE resolved thermolysin-treated or -untreated envelope membranes (50 μg of protein). The position of Tic and Toc components and pS-1 are indicated to the left of the figure. (B) Fluorograph of SDS-PAGE resolved immunoaffinity eluates of anti-Tic22 IgG Sepharose and preimmune IgG Sepharose.

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)

Immunoaffinity purification of [125I]Tic22 with anti-Tic22 IgG Sepharose. Intact chloroplasts were cross-linked to  pS-1 in the presence of 0.1 mM ATP and GTP. After import, the  chloroplasts were treated in the absence or presence of 100 μg/ml  thermolysin for 30 min on ice to remove bound pS-1, and chloroplast were subfractionated to yield a total envelope membrane  fraction. Envelope membranes (80 μg of protein) from cross-linked chloroplasts were dissolved under denaturing conditions,  and applied to anti-Tic22 Sepharose (α-Tic22) or preimmune IgG  Sepharose (PI) as indicated. (A) Fluorograph of SDS-PAGE resolved thermolysin-treated or -untreated envelope membranes  (50 μg of protein). The position of Tic and Toc components and  pS-1 are indicated to the left of the figure. (B) Fluorograph of  SDS-PAGE resolved immunoaffinity eluates of anti-Tic22 IgG  Sepharose and preimmune IgG Sepharose.
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Figure 2: Immunoaffinity purification of [125I]Tic22 with anti-Tic22 IgG Sepharose. Intact chloroplasts were cross-linked to pS-1 in the presence of 0.1 mM ATP and GTP. After import, the chloroplasts were treated in the absence or presence of 100 μg/ml thermolysin for 30 min on ice to remove bound pS-1, and chloroplast were subfractionated to yield a total envelope membrane fraction. Envelope membranes (80 μg of protein) from cross-linked chloroplasts were dissolved under denaturing conditions, and applied to anti-Tic22 Sepharose (α-Tic22) or preimmune IgG Sepharose (PI) as indicated. (A) Fluorograph of SDS-PAGE resolved thermolysin-treated or -untreated envelope membranes (50 μg of protein). The position of Tic and Toc components and pS-1 are indicated to the left of the figure. (B) Fluorograph of SDS-PAGE resolved immunoaffinity eluates of anti-Tic22 IgG Sepharose and preimmune IgG Sepharose.
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