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Ribosome nascent chain complexes of the chloroplast-encoded cytochrome b6 thylakoid membrane protein interact with cpSRP54 but not with cpSecY.

Piskozub M, Króliczewska B, Króliczewski J - J. Bioenerg. Biomembr. (2015)

Bottom Line: We showed that the cytochrome b6 nascent polypeptide complex is tightly associated with ribosomes and that the translation of cytochrome b6 was discontinuous.It was also found that cpSecY was not in the vicinity of cytochrome b6 intermediates during the elongation process and does not act with mature cytochrome b6 after translation.Using the approach of cross-linking during elongation of the cytochrome b6 protein, we showed that cpSRP54 interacts strongly with the elongating nascent chain.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Biotechnology, University of Wrocław, Fryderyka Joliot-Curie 14a, 50-383, Wroclaw, Poland.

ABSTRACT
We analysed the interplay between the cpSecY, cpSRP54 and the chloroplast-encoded cytochrome b6 via isolation of chloroplast ribosome nascent chain complexes and the use of cross-linking factors, antibodies and mass spectroscopy analyses. We showed that the cytochrome b6 nascent polypeptide complex is tightly associated with ribosomes and that the translation of cytochrome b6 was discontinuous. The causes of ribosome pausing and the functional significance of this phenomenon may be related to proper protein folding, insertion into thylakoid membranes and the association of cofactors during this process. It was also found that cpSecY was not in the vicinity of cytochrome b6 intermediates during the elongation process and does not act with mature cytochrome b6 after translation. Using the approach of cross-linking during elongation of the cytochrome b6 protein, we showed that cpSRP54 interacts strongly with the elongating nascent chain.

No MeSH data available.


Related in: MedlinePlus

Western blot analysis of RNC–D1 complexes and RNC–cytochrome b6 complexes. a D1 nascent chain was isolated and a homo-bifunctional cross-linker (BMH) was added. The samples were immunoprecipitated with antibody against D1 protein, solubilized with SDS, and subjected to further immunoprecipitation with anti-cpSecY. The D1 elongation intermediates as well as the mature D1 are indicated by arrows. b Western blot analysis of the cross-linking (BMH) of isolated RNCs followed by immunoprecipitation with antiserum directed against the N-terminal part of cytochrome b6. Gel bands subjected to PMF are indicated by arrows. The bands marked with filled stars contain cytochrome b6 intermediates. c Western blot analysis of the cross-linking of isolated RNC-cytochrome b6 complexes immunoprecipitated with antibody directed against the N-terminal part of cytochrome b6, solubilized with SDS and subjected to further immunoprecipitation with anti-cpSecY. A very weak signal marked by triangle was observed. MS analysis of corresponding gel bands from SDS-PAGE was performed
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Fig3: Western blot analysis of RNC–D1 complexes and RNC–cytochrome b6 complexes. a D1 nascent chain was isolated and a homo-bifunctional cross-linker (BMH) was added. The samples were immunoprecipitated with antibody against D1 protein, solubilized with SDS, and subjected to further immunoprecipitation with anti-cpSecY. The D1 elongation intermediates as well as the mature D1 are indicated by arrows. b Western blot analysis of the cross-linking (BMH) of isolated RNCs followed by immunoprecipitation with antiserum directed against the N-terminal part of cytochrome b6. Gel bands subjected to PMF are indicated by arrows. The bands marked with filled stars contain cytochrome b6 intermediates. c Western blot analysis of the cross-linking of isolated RNC-cytochrome b6 complexes immunoprecipitated with antibody directed against the N-terminal part of cytochrome b6, solubilized with SDS and subjected to further immunoprecipitation with anti-cpSecY. A very weak signal marked by triangle was observed. MS analysis of corresponding gel bands from SDS-PAGE was performed

Mentions: To identify chloroplast proteins interacting with the cytochrome b6RNCs, a cross-linking approach, followed by immunoprecipitation with specific antibodies (an antibody against cytochrome b6, D1, or cpSecY) was applied. Freshly prepared RNCs were purified from intact pea chloroplast and then incubated with BMH. Cross-linked products with BMH were immunoprecipitated with appropriate antiserum (against D1 protein or against cytochrome b6) after denaturing with SDS. The precipitated products were subjected to further immunoprecipitation with anti-cpSecY. After the second immunoprecipitation, precipitated products were separated on Tricine PAGE and analyzed with Western blot (Fig. 3). Initially, examination of the interaction of soluble stromal proteins with the D1 nascent chain emerging out of the ribosome tunnel was performed. Full-length D1 protein and intermediates synthesized from psbA transcripts in the pea chloroplast were observed and developed stable and D1 was co-immunoprecipitated with cpSecY (Fig. 3a).Fig. 3


Ribosome nascent chain complexes of the chloroplast-encoded cytochrome b6 thylakoid membrane protein interact with cpSRP54 but not with cpSecY.

Piskozub M, Króliczewska B, Króliczewski J - J. Bioenerg. Biomembr. (2015)

Western blot analysis of RNC–D1 complexes and RNC–cytochrome b6 complexes. a D1 nascent chain was isolated and a homo-bifunctional cross-linker (BMH) was added. The samples were immunoprecipitated with antibody against D1 protein, solubilized with SDS, and subjected to further immunoprecipitation with anti-cpSecY. The D1 elongation intermediates as well as the mature D1 are indicated by arrows. b Western blot analysis of the cross-linking (BMH) of isolated RNCs followed by immunoprecipitation with antiserum directed against the N-terminal part of cytochrome b6. Gel bands subjected to PMF are indicated by arrows. The bands marked with filled stars contain cytochrome b6 intermediates. c Western blot analysis of the cross-linking of isolated RNC-cytochrome b6 complexes immunoprecipitated with antibody directed against the N-terminal part of cytochrome b6, solubilized with SDS and subjected to further immunoprecipitation with anti-cpSecY. A very weak signal marked by triangle was observed. MS analysis of corresponding gel bands from SDS-PAGE was performed
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4555342&req=5

Fig3: Western blot analysis of RNC–D1 complexes and RNC–cytochrome b6 complexes. a D1 nascent chain was isolated and a homo-bifunctional cross-linker (BMH) was added. The samples were immunoprecipitated with antibody against D1 protein, solubilized with SDS, and subjected to further immunoprecipitation with anti-cpSecY. The D1 elongation intermediates as well as the mature D1 are indicated by arrows. b Western blot analysis of the cross-linking (BMH) of isolated RNCs followed by immunoprecipitation with antiserum directed against the N-terminal part of cytochrome b6. Gel bands subjected to PMF are indicated by arrows. The bands marked with filled stars contain cytochrome b6 intermediates. c Western blot analysis of the cross-linking of isolated RNC-cytochrome b6 complexes immunoprecipitated with antibody directed against the N-terminal part of cytochrome b6, solubilized with SDS and subjected to further immunoprecipitation with anti-cpSecY. A very weak signal marked by triangle was observed. MS analysis of corresponding gel bands from SDS-PAGE was performed
Mentions: To identify chloroplast proteins interacting with the cytochrome b6RNCs, a cross-linking approach, followed by immunoprecipitation with specific antibodies (an antibody against cytochrome b6, D1, or cpSecY) was applied. Freshly prepared RNCs were purified from intact pea chloroplast and then incubated with BMH. Cross-linked products with BMH were immunoprecipitated with appropriate antiserum (against D1 protein or against cytochrome b6) after denaturing with SDS. The precipitated products were subjected to further immunoprecipitation with anti-cpSecY. After the second immunoprecipitation, precipitated products were separated on Tricine PAGE and analyzed with Western blot (Fig. 3). Initially, examination of the interaction of soluble stromal proteins with the D1 nascent chain emerging out of the ribosome tunnel was performed. Full-length D1 protein and intermediates synthesized from psbA transcripts in the pea chloroplast were observed and developed stable and D1 was co-immunoprecipitated with cpSecY (Fig. 3a).Fig. 3

Bottom Line: We showed that the cytochrome b6 nascent polypeptide complex is tightly associated with ribosomes and that the translation of cytochrome b6 was discontinuous.It was also found that cpSecY was not in the vicinity of cytochrome b6 intermediates during the elongation process and does not act with mature cytochrome b6 after translation.Using the approach of cross-linking during elongation of the cytochrome b6 protein, we showed that cpSRP54 interacts strongly with the elongating nascent chain.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Biotechnology, University of Wrocław, Fryderyka Joliot-Curie 14a, 50-383, Wroclaw, Poland.

ABSTRACT
We analysed the interplay between the cpSecY, cpSRP54 and the chloroplast-encoded cytochrome b6 via isolation of chloroplast ribosome nascent chain complexes and the use of cross-linking factors, antibodies and mass spectroscopy analyses. We showed that the cytochrome b6 nascent polypeptide complex is tightly associated with ribosomes and that the translation of cytochrome b6 was discontinuous. The causes of ribosome pausing and the functional significance of this phenomenon may be related to proper protein folding, insertion into thylakoid membranes and the association of cofactors during this process. It was also found that cpSecY was not in the vicinity of cytochrome b6 intermediates during the elongation process and does not act with mature cytochrome b6 after translation. Using the approach of cross-linking during elongation of the cytochrome b6 protein, we showed that cpSRP54 interacts strongly with the elongating nascent chain.

No MeSH data available.


Related in: MedlinePlus