Abstract
The mechanisms of targeting and insertion of chloroplast-encoded thylakoid membrane proteins are poorly understood. In this study, we have used a translation system isolated from chloroplasts to begin to investigate these mechanisms. The bacterial membrane protein leader peptidase (Lep) was used as a model protein because its targeting and insertion mechanisms are well understood for Escherichia coli and for the endoplasmic reticulum. Lep could thus provide insight into the functional homologies between the different membrane systems. Lep was efficiently expressed in the chloroplast translation system, and the protein could be inserted into thylakoid membranes with the same topology as in E. coli cytoplasmic membranes, following the positive-inside rule. Insertion of Lep into the thylakoid membrane was stimulated by the trans-thylakoid proton gradient and was strongly inhibited by azide, suggesting a requirement for SecA activity. Insertion most likely occurred in a cotranslational manner, because insertion could only be observed if thylakoid membranes were present during translation reactions but not when thylakoid membranes were added after translation reactions were terminated. To halt the elongation process at different stages, we translated truncated Lep mRNAs without a stop codon, resulting in the formation of stable ribosome nascent chain complexes. These complexes showed a strong, salt-resistant affinity for the thylakoid membrane, implying a functional interaction of the ribosome with the membrane and supporting a cotranslational insertion mechanism for Lep. Our study supports a functional homology for the insertion of Lep into the thylakoid membrane and the E. coli cytoplasmic membrane.
Highlights
Little is known about the mechanisms of targeting and insertion of the chloroplast-encoded thylakoid membrane proteins, despite their importance in chloroplast function (Jagendorf and Michaels, 1990; Kuras et al, 1995; van Wijk et al, 1995; Rochaix, 1996; Choquet et al, 1998)
When the chloroplast translation system isolated from tobacco leaves was programmed with this mRNA, a high translation yield of full-length leader peptidase (Lep) was observed without the problem of premature termination (Figure 2A, left)
We have addressed the mechanisms of protein insertion into the thylakoid membrane, by using a chloroplast translation system
Summary
Little is known about the mechanisms of targeting and insertion of the chloroplast-encoded thylakoid membrane proteins, despite their importance in chloroplast function (Jagendorf and Michaels, 1990; Kuras et al, 1995; van Wijk et al, 1995; Rochaix, 1996; Choquet et al, 1998). Based on a number of observations in chloroplasts, such as run-off translations of thylakoids with bound ribosomes (rough thylakoids) and detection of translation intermediates in the membrane, it can be postulated that insertion of the polytopic chloroplast–encoded membrane proteins occurs cotranslationally (Margulies and Michaels, 1975; Herrin and Michaels, 1985; Klein et al, 1988; van Wijk et al, 1996). To reconstitute this targeting and insertion process, a chloroplast in vitro initiation/translation system is required in which exogenous transcripts can be accurately translated.
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