Merck
  • Home
  • Search Results
  • In vitro reconstitution of co-translational D1 insertion reveals a role of the cpSec-Alb3 translocase and Vipp1 in photosystem II biogenesis.

In vitro reconstitution of co-translational D1 insertion reveals a role of the cpSec-Alb3 translocase and Vipp1 in photosystem II biogenesis.

The Biochemical journal (2015-03-25)
Björn Walter, Athina Hristou, Marc M Nowaczyk, Danja Schünemann
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Sucrose, ≥99.5%
Sigma-Aldrich
Potassium acetate, BioXtra, ≥99.0%
Sigma-Aldrich
Potassium acetate, meets USP testing specifications
Sigma-Aldrich
Sucrose, meets USP testing specifications
Sigma-Aldrich
Sucrose, BioXtra, ≥99.5% (GC)
Sigma-Aldrich
Sucrose, ACS reagent
Sigma-Aldrich
Potassium acetate, for molecular biology, ≥99.0%
Sigma-Aldrich
Sucrose, Grade II, suitable for plant cell culture
Sigma-Aldrich
Sucrose, for molecular biology, ≥99.5% (GC)
Sigma-Aldrich
Sucrose, Grade I, suitable for plant cell culture
Sigma-Aldrich
Sucrose, BioReagent, suitable for cell culture, suitable for insect cell culture, ≥99.5% (GC)
Sigma-Aldrich
Chloramphenicol, BioReagent, suitable for plant cell culture
Sigma-Aldrich
Chloramphenicol, γ-irradiated
Sigma-Aldrich
Chloramphenicol, meets USP testing specifications
Sigma-Aldrich
Chloramphenicol, ≥98% (HPLC)
Sigma-Aldrich
Creatine, anhydrous
Sigma-Aldrich
Sucrose, ≥99.5% (GC)
Sigma-Aldrich
Potassium acetate, 98%
Sigma-Aldrich
Potassium acetate, BioUltra, for molecular biology, ≥99.0% (NT)
Sigma-Aldrich
4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride, ≥97.0% (HPLC)
Sigma-Aldrich
Potassium acetate, 99.98% trace metals basis
Sigma-Aldrich
Sucrose, puriss., meets analytical specification of Ph. Eur., BP, NF
Sigma-Aldrich
Sucrose, BioUltra, for molecular biology, ≥99.5% (HPLC)
Sigma-Aldrich
Sodium chloride solution, 5 M
Sigma-Aldrich
D-Sorbitol, BioXtra, ≥98%
Sigma-Aldrich
D-Sorbitol, ≥98%
Sigma-Aldrich
Sodium chloride solution, 5 M in H2O, BioReagent, for molecular biology, suitable for cell culture
Sigma-Aldrich
D-Sorbitol, for molecular biology, ≥98%
Sigma-Aldrich
D-Sorbitol, BioReagent, suitable for cell culture, suitable for plant cell culture
Sigma-Aldrich
D-Sorbitol, FCC, FG