Limits...
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.


Cross-linked RNC complexes isolated from intact chloroplasts. Purified ribosome-nascent chain complexes bearing cytochrome b6 were cross-linked with interacting components using BMH. a SDS-PAGE of cross-linked RNC complexes. Lane 1, molecular mass standards; lane 2, RNC preparation from pea chloroplast. b 1-D BN-PAGE gel of cross-linked RNC complexes stained by CBB R-250. Gel bands subjected to PMF are indicated by filled circle.c Western blot analysis of the gel prepared as in (b), detection by antibody against NH2-terminus of cytochrome b6. The arrowhead indicates the bands that contained cross-linked cytochrome b6
© Copyright Policy - OpenAccess
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4555342&req=5

Fig1: Cross-linked RNC complexes isolated from intact chloroplasts. Purified ribosome-nascent chain complexes bearing cytochrome b6 were cross-linked with interacting components using BMH. a SDS-PAGE of cross-linked RNC complexes. Lane 1, molecular mass standards; lane 2, RNC preparation from pea chloroplast. b 1-D BN-PAGE gel of cross-linked RNC complexes stained by CBB R-250. Gel bands subjected to PMF are indicated by filled circle.c Western blot analysis of the gel prepared as in (b), detection by antibody against NH2-terminus of cytochrome b6. The arrowhead indicates the bands that contained cross-linked cytochrome b6

Mentions: To follow cytochrome b6 translation elongation and the concomitant translocation and insertion into the thylakoid membrane, we isolated RNC complexes (Fig. 1a, lane 2, SDS-PAGE of RNC complexes) and next cross-linked via BMH, and then quantitatively immunoprecipitated the cytochrome b6 nascent chains with an excess of antibody against N-terminal residues of cytochrome b6.Fig. 1


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)

Cross-linked RNC complexes isolated from intact chloroplasts. Purified ribosome-nascent chain complexes bearing cytochrome b6 were cross-linked with interacting components using BMH. a SDS-PAGE of cross-linked RNC complexes. Lane 1, molecular mass standards; lane 2, RNC preparation from pea chloroplast. b 1-D BN-PAGE gel of cross-linked RNC complexes stained by CBB R-250. Gel bands subjected to PMF are indicated by filled circle.c Western blot analysis of the gel prepared as in (b), detection by antibody against NH2-terminus of cytochrome b6. The arrowhead indicates the bands that contained cross-linked cytochrome b6
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4555342&req=5

Fig1: Cross-linked RNC complexes isolated from intact chloroplasts. Purified ribosome-nascent chain complexes bearing cytochrome b6 were cross-linked with interacting components using BMH. a SDS-PAGE of cross-linked RNC complexes. Lane 1, molecular mass standards; lane 2, RNC preparation from pea chloroplast. b 1-D BN-PAGE gel of cross-linked RNC complexes stained by CBB R-250. Gel bands subjected to PMF are indicated by filled circle.c Western blot analysis of the gel prepared as in (b), detection by antibody against NH2-terminus of cytochrome b6. The arrowhead indicates the bands that contained cross-linked cytochrome b6
Mentions: To follow cytochrome b6 translation elongation and the concomitant translocation and insertion into the thylakoid membrane, we isolated RNC complexes (Fig. 1a, lane 2, SDS-PAGE of RNC complexes) and next cross-linked via BMH, and then quantitatively immunoprecipitated the cytochrome b6 nascent chains with an excess of antibody against N-terminal residues of cytochrome b6.Fig. 1

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.