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S Leu¹, R Bolli, L Mendiola-Morgenthaler

¹Istitut für Biochemie der Universität, Freiestrasse 3, CH-3012, Bern, Switzerland.

|November 22, 2013

Abstract:

Starting from isolated chloroplasts of the Chlamydomonas reinhardii cw 15 mutant, several mRNA-containing chloroplast subfractions, i.e. thylakoid-bound polysomes, detached polysomes or isolated RNA, were prepared and incubated in homologous and heterologous translation systems. In the reticulocyte lysate these fractions gave rise to strikingly different product patterns. A most prominent difference concerned the in-vivo rapidly labelled 32,000-dalton thylakoid polypeptide. Neither this membrane protein nor its 34,000-dalton precursor was formed when membrane-containing or free polysomes were translated, while the 34,000-dalton precursor was a main product of the RNA isolated from the same membranes. The influence of thylakoid membranes during translation was also observed in homologous translation systems with lysed chloroplasts supplemented with ATP. Membrane and soluble fractions, when translated separately, yielded product patterns which differed from each other, although the RNAs extracted from the respective fractions gave the same product patterns when translated in reticulocyte lysate; the latter included a soluble protein, the large subunit of ribulose-1,5-bisphosphate carboxylase, and a membrane protein, the 34,000-dalton precursor of the 32,000-dalton membrane protein, as major labelled translation products. These results point to a regulatory role of thylakoid membranes in the expression of chloroplast mRNA and argue against compartmentation of the chloroplast mRNAs between the soluble and membrane fractions.

Insights

Thylakoid membranes regulate chloroplast gene expression in Chlamydomonas reinhardii. Membrane association influences protein synthesis, affecting the production of key thylakoid polypeptides.

Area of Science:

* Chloroplast biology
* Molecular genetics
* Protein synthesis

Background:

* Chloroplasts contain their own genetic material and protein synthesis machinery.
* The expression of chloroplast genes is complex and not fully understood.
* Thylakoid membranes are crucial for photosynthesis and may play a role in gene regulation.

Purpose of the Study:

* To investigate the role of thylakoid membranes in regulating chloroplast mRNA translation.
* To determine if chloroplast mRNA is compartmentalized between soluble and membrane fractions.
* To analyze the influence of membrane association on protein product patterns.

Main Methods:

* Isolated chloroplasts from Chlamydomonas reinhardii cw 15 mutant.
* Preparation of thylakoid-bound polysomes, detached polysomes, and isolated RNA.
* In vitro translation assays using homologous (lysed chloroplasts) and heterologous (reticulocyte lysate) systems.

Main Results:

* Different mRNA fractions yielded distinct protein patterns in the reticulocyte lysate system.
* The 32,000-dalton thylakoid polypeptide and its precursor were not synthesized by membrane-containing or free polysomes.
* RNA isolated from membranes produced the 34,000-dalton precursor, suggesting membrane influence during translation.

Conclusions:

* Thylakoid membranes play a regulatory role in chloroplast gene expression.
* Evidence suggests against the compartmentalization of chloroplast mRNAs into soluble and membrane fractions.
* Membrane association influences the translation of specific chloroplast mRNAs.

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