RRC ID 38625
著者 Walter B, Hristou A, Nowaczyk MM, Schünemann D.
タイトル In vitro reconstitution of co-translational D1 insertion reveals a role of the cpSec-Alb3 translocase and Vipp1 in photosystem II biogenesis.
ジャーナル Biochem J
Abstract Photosystem II (PS II) is a multi-subunit complex localized in the thylakoid membrane that performs the light-dependent photosynthetic charge separation. The PS II reaction centre comprises, among others, the D1 protein. De novo synthesis and repair of PS II require efficient mechanisms for transport and insertion of plastid encoded D1 into the thylakoid membrane. To elucidate the process of D1 insertion, we used an in vitro translation system derived from pea chloroplasts to reconstitute the D1 insertion. Thereby, truncated D1 encoding psbA mRNAs lacking a stop codon were translated in the presence of thylakoid membranes and the translation was stalled by addition of chloramphenicol. The generated ribosome nascent chain complexes (RNCs) were tightly associated with the thylakoids. Subsequently, these D1 insertion intermediates were enriched from solubilized thylakoids by sucrose cushion centrifugation. Immunological analyses demonstrated the presence of the cpSec translocase, Alb3, cpFtsY, cpSRP54 and Vipp1 (vesicle-inducing protein in plastids 1) in the enriched D1 insertion intermediates. A complex formation between cpSecY, Alb3, cpFtsY and Vipp1 in thylakoid membranes was shown by gel filtration chromatography, BN (Blue Native)/SDS-PAGE and co-immunoprecipitation experiments. Furthermore, a stimulating effect of recombinant Vipp1 on the formation of a D1 insertion intermediate was observed in vitro. These results suggest a co-operative function of these proteins in D1 insertion.
巻・号 468(2)
ページ 315-24
公開日 2015-6-1
DOI 10.1042/BJ20141425
PII BJ20141425
PMID 25803492
MeSH Arabidopsis / metabolism Arabidopsis Proteins / metabolism* Chloroplasts / metabolism* Chromatography, Gel Immunoprecipitation In Vitro Techniques Intracellular Membranes / metabolism* Light Membrane Proteins / metabolism* Peas / metabolism Photosystem II Protein Complex / biosynthesis* Plant Proteins / metabolism* Protein Biosynthesis Thylakoids / metabolism*
IF 4.097
引用数 22
WOS 分野 BIOCHEMISTRY & MOLECULAR BIOLOGY
リソース情報
シロイヌナズナ / 植物培養細胞・遺伝子 pda02786