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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.


Western blot analysis of (a) D1 protein translation elongation in intact chloroplasts. The thylakoid membrane-bound RNCs were solubilized with SDS, and D1 elongation intermediates were immunoprecipitated with an excess of specific N-terminal D1 antibody. b Western blot of several cytochrome b6 protein translation elongation intermediates in intact chloroplasts. The thylakoid membrane-bound RNCs were solubilized with SDS, and cytochrome b6 elongation intermediates were immunoprecipitated with an excess of N-terminal b6 antibody. Precipitated RNCs were separated by SDS-PAGE and visualized by Western blot. The D1 elongation intermediates are indicated as well as the mature (D1) and precursor (pD1) forms. The cytochrome b6 elongation intermediates of 18, 14, and 10 kDa as well as the mature (cytb6) forms are indicated. The molecular mass (kDa) of D1 and cytochrome b6 intermediates and their identification was carried out using mass spectrometry (Table SP1)
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Fig2: Western blot analysis of (a) D1 protein translation elongation in intact chloroplasts. The thylakoid membrane-bound RNCs were solubilized with SDS, and D1 elongation intermediates were immunoprecipitated with an excess of specific N-terminal D1 antibody. b Western blot of several cytochrome b6 protein translation elongation intermediates in intact chloroplasts. The thylakoid membrane-bound RNCs were solubilized with SDS, and cytochrome b6 elongation intermediates were immunoprecipitated with an excess of N-terminal b6 antibody. Precipitated RNCs were separated by SDS-PAGE and visualized by Western blot. The D1 elongation intermediates are indicated as well as the mature (D1) and precursor (pD1) forms. The cytochrome b6 elongation intermediates of 18, 14, and 10 kDa as well as the mature (cytb6) forms are indicated. The molecular mass (kDa) of D1 and cytochrome b6 intermediates and their identification was carried out using mass spectrometry (Table SP1)

Mentions: The control protein in the experiment was the D1 protein. The D1 protein has the highest turnover rate of the known thylakoid membrane proteins and in vivo it is constantly synthesized and inserted into the mature thylakoid membrane system (Zhang et al. 2001). Data from in vivo and cell-free experiments suggested that synthesis of proteins may proceed discontinuously due to translational pausing. During elongation of the D1 protein, ribosomes pause at several distinct sites, generating pausing intermediates 17–25 kDa (Zhang et al. 1999, 2000). The D1 intermediates were precipitated with an excess of D1 antibody (Zhang et al. 2001). We found (Fig. 2a) that several of the Dl translation intermediates co-sediment with polysomes, and this results was in accordance with previous investigations (Kim et al. 1991; Zhang et al. 1999).Fig. 2


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 (a) D1 protein translation elongation in intact chloroplasts. The thylakoid membrane-bound RNCs were solubilized with SDS, and D1 elongation intermediates were immunoprecipitated with an excess of specific N-terminal D1 antibody. b Western blot of several cytochrome b6 protein translation elongation intermediates in intact chloroplasts. The thylakoid membrane-bound RNCs were solubilized with SDS, and cytochrome b6 elongation intermediates were immunoprecipitated with an excess of N-terminal b6 antibody. Precipitated RNCs were separated by SDS-PAGE and visualized by Western blot. The D1 elongation intermediates are indicated as well as the mature (D1) and precursor (pD1) forms. The cytochrome b6 elongation intermediates of 18, 14, and 10 kDa as well as the mature (cytb6) forms are indicated. The molecular mass (kDa) of D1 and cytochrome b6 intermediates and their identification was carried out using mass spectrometry (Table SP1)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig2: Western blot analysis of (a) D1 protein translation elongation in intact chloroplasts. The thylakoid membrane-bound RNCs were solubilized with SDS, and D1 elongation intermediates were immunoprecipitated with an excess of specific N-terminal D1 antibody. b Western blot of several cytochrome b6 protein translation elongation intermediates in intact chloroplasts. The thylakoid membrane-bound RNCs were solubilized with SDS, and cytochrome b6 elongation intermediates were immunoprecipitated with an excess of N-terminal b6 antibody. Precipitated RNCs were separated by SDS-PAGE and visualized by Western blot. The D1 elongation intermediates are indicated as well as the mature (D1) and precursor (pD1) forms. The cytochrome b6 elongation intermediates of 18, 14, and 10 kDa as well as the mature (cytb6) forms are indicated. The molecular mass (kDa) of D1 and cytochrome b6 intermediates and their identification was carried out using mass spectrometry (Table SP1)
Mentions: The control protein in the experiment was the D1 protein. The D1 protein has the highest turnover rate of the known thylakoid membrane proteins and in vivo it is constantly synthesized and inserted into the mature thylakoid membrane system (Zhang et al. 2001). Data from in vivo and cell-free experiments suggested that synthesis of proteins may proceed discontinuously due to translational pausing. During elongation of the D1 protein, ribosomes pause at several distinct sites, generating pausing intermediates 17–25 kDa (Zhang et al. 1999, 2000). The D1 intermediates were precipitated with an excess of D1 antibody (Zhang et al. 2001). We found (Fig. 2a) that several of the Dl translation intermediates co-sediment with polysomes, and this results was in accordance with previous investigations (Kim et al. 1991; Zhang et al. 1999).Fig. 2

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.