Synthesis and turnover of photosystem II reaction center protein D1. Ribosome pausing increases during chloroplast development.

J Kim,P.G Klein,J.E Mullet

doi:10.1016/s0021-9258(17)32397-9

Abstract

The chloroplast photosystem II reaction center protein D1 contains five membrane-spanning helices and binds chlorophyll, carotenoid, quinone, iron, and probably manganese. Turnover of pulse-labeled D1 in isolated plastids was found to involve cleavage between helix IV and helix V, which releases a 23-kDa N-terminal peptide and two C-terminal peptides of 10 and 8 kDa. Ribosomes pause at specific sites during translation of D1, which results in the accumulation of D1 translation intermediates. Pulse-labeling assays followed by polysome isolation and immunoprecipitation identified paused D1 translation intermediates of 9, 12.5, 15-18, 20, 21, 24, and 28-32 kDa. Ribosome pausing was not altered when dark-grown seedlings were illuminated for up to 1 h, even though this treatment stimulated accumulation of chlorophyll and D1. However, illumination of plants for 16-72 h resulted in increased ribosome pausing and the build-up of D1 translation intermediates. We hypothesize that ribosome pausing during synthesis of D1 improves the efficiency of chlorophyll binding of D1 nascent chains and enhances accumulation of D1 in mature chloroplasts, which have reduced rates of chlorophyll biosynthesis.

Highlights

The chloroplast photosystem 11 reaction center pro- cofactors associate with the individual subunits of the reaction tein D l contains five membrane-spanning helices and center prior to protein dimerization
Illuminationof plants somes (Klein et al, 1988333 Mullet et al, 1990).When plastids for 16-72 h resulted in increased ribosome pausing andfrom dark-grown plants are pulse-labeled, D l translation inthe build-up oDfl translation intermediatesW. e hypoth- termediates and degradation products are radiolabeled, but esize that ribosome pausing during synthesiosf D l im- little full-length D l accumulates (Mullet et at., 1990).Accumuproves the efficiency of chlorophyll binding toD l nas- lation of D l is dependent on light-induced chlorophyll synthecent chains and enhances accumulatoiof nD l in mature sis (Eichacker et al, 1990; Kleinand Mullet, 1986, 1987; Klein chloroplasts, which have reduced rates of chlorophyll et al, 1988a)
In this study we further investigated the relationship between ribosome pausing, D l translation intermediates,and D l turnover

Summary

RIBOSOME PAUSING INCREASES DURING CHLOROPLAST DEVELOPMENT"

(Received for publication, January 24, 1994, and in revised form, March 15, 1994). From the Department of Biochemistry and Biophysics, TexasA & M University, College Station, Ikxas 77843-2128 and the. Ribosomes pause at specificsites during translation of cofactors, associate with each other and other photosystem I1. When dark-grown seedlings were illuminated for 1up tDoark-grown plants lack D l (Klein and Mullet, 1987), even h, even though this treatment stimulated accumulatiotnhough psbA mRNA accumulates and is associated with polyof chlorophyll andDl. illuminationof plants somes (Klein et al, 1988333 Mullet et al, 1990).When plastids for 16-72 h resulted in increased ribosome pausing andfrom dark-grown plants are pulse-labeled, D l translation inthe build-up oDfl translation intermediatesW. Some of the cofactors associated with the photosynthetic (1994)).Analysis of D l turnover revealed degradation products reaction centers are positioned within the heterodimer (Deisen- of 23, 17, 16.5, 14, and 8-12 kDa and a protease cleavage site hofer and Michel, 1989).it seems likely that these between helix IV and helix V D l degradation products and D l translation intermediates were identified providing a description of D l synthesis and turnover in barleychloroplasts

MATERIALSAND METHODS

RESULTS

DISCUSSION