Isolation of cDNA clones for fourteen nuclear-encoded thylakoid membrane proteins

Tittgen, J.; Hermans, J.; Steppuhn, Johannes; Jansen, Thomas; Jansson, Christer; Andersson, Bertil; Nechushtai, Rachel; Nelson, Nathan; Herrmann, Reinhold G.

doi:10.1007/bf00425507

Cited by 95 publications

(77 citation statements)

References 31 publications

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“…First, both polypeptides are synthesized in the cytoplasm in a precursor form, which is converted to the mature polypeptide upon import into the chloroplast [4,33,341. No intermediate processing step was found, as is the case for lumentargetted polypeptides.…”

Section: Discussionmentioning

confidence: 99%

Purification and membrane topology of PSI‐D and PSI‐E, two subunits of the photosystem I reaction center

Lagoutte

Valloni

1992

European Journal of Biochemistry

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Structural studies have been conducted on polypeptides PSI-D and PSI-E, whch are extrinsic but firmly bound to the photosystem I reaction center. These subunits are predicted to be involved in the correct interaction with soluble electron acceptor(s), like ferredoxin. We designed an original method to extract both polypeptides directly from thylakoid membranes and to purify them: a stepwise extraction with NaSCN followed by size fractionation and reverse-phase HPLC. Investigation of the in situ topology of PSI-D and PSI-E was undertaken using monoclonal antibody binding, controlled proteolysis, peptide sequencing and electron microscopy. The precise identification of numerous proteolytic sites indicates that the entire N-terminal regions of PSI-E (up to Glu15) and PSI-D (up to LyslS) are exposed to the medium. Partial mapping of the exposed epitopes was possible using purified fragments of each polypeptide. In the case of PSI-E, this mapping confirmed the accessibility of the N-terminal part, and suggested the need for another exposed sequence, probably located after Met39 in the second half of the protein. For PSI-D, this mapping revealed that the sequence between Met74 and Metl40, including the most basic amino acid clusters, is also partly accessible. These experiments provide the first detailed informations, although still partial, on the topology of these polypeptides. They give a preliminary basis for hypotheses concerning the sites of interaction with the soluble counterparts.

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Section: Discussionmentioning

confidence: 99%

Purification and membrane topology of PSI‐D and PSI‐E, two subunits of the photosystem I reaction center

Lagoutte

Valloni

1992

European Journal of Biochemistry

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“…Screening of the cDNA library, constructed from polyA+-RNA isolated from etiolated spinach cotyledone after 16 h illumination, was done as previously described [16].…”

Section: Construction Of Phage-based Expression Libraries Library Scmentioning

confidence: 99%

The 64 kDa polypeptide of spinach may not be the LHCII kinase, but a lumen‐located polyphenol oxidase

et al. 1995

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“…gtll expression library prepared from polyadenylated RNA from N. tabncltm Samsun NN (Palomares et al, 1991 was screened with monospecific polyclonal antisera raised against the spinach 33-kDa and 23-kDa polypeptides of the water-oxidation complex (Tittgen et al, 1986). Positive phage were rescreened, the DNA isolated, digested with EcoRI, the inserts cloned into Bluescript pBSC' (Stratagene) and sequenced following standard techniques (Sambrook et al, 1989).…”

Section: Isolation Of Cdna Clonesmentioning

confidence: 99%

Post‐transcriptional and post‐translational regulatory steps are crucial in controlling the appearance and stability of thylakoid polypeptides during the transition of etiolated tobacco seedlings to white light

Palomares

Herrmann

Oelmüller

1993

European Journal of Biochemistry

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We have investigated the expression of nuclear-encoded chloroplast proteins that are not associated with chlorophyll (the lumenal 33-kDa and 23-kDa polypeptides of the oxygen-evolving system of photosystem 11, plastocyanin and the Rieske Fe/S protein) by comparing mRNA-accumulation rates with those of the corresponding proteins during illumination of etiolated tobacco seedlings. Using subcellular fractionation, pulsekhase, Northern and Western techniques, we found that the biogenesis and stability of these proteins are regulated both translationally, as well as post-translationally, including the efficiency of mRNA uptake into polysomes, processes that operate between translation and assembly or monitor the status (soluble and membrane-attached) of a terminally processed polypeptide. Polypeptide synthesis is generally not limited by mRNA amounts. For instance, steady-state transcript levels may increase 10-fold during illumination, while those associated with polycomes increase only 2-3-fold without measurable influence on the rate of protein synthesis. The 23-kDa and Rieske polypeptides are predominantely membrane associated, but plastocyanin and the 33-kDa polypeptide are distributed among both soluble and membrane-associated protein fractions. Plastocyanin appears to be comparably stable in both forms. However, for the 33-kDa polypcptide, only the membrane-attached form is stable (> 8 h) and only this pool increases upon illumination. Its soluble form is rapidly degraded with a half-life of approximately 1 h under the chosen conditions. Our findings probably reflect part of a more general regulatory principle operating in the differentiation and maintenance of subcellular structure.Thylakoid membranes of higher plants consist of approximately 70 polypcptide species, the majority of which are assembled in five major multisubunit complexes, the photosystems I and 11, the cytochrome complex, the ATP synthase and the NAD(P)H dehydrogenase. Most of the genes that code for these polypeptides have been isolated during the past decadc (summarized in Hallick, 1989, andHemnann et al., 1991); approximately half of them originate in plastids, the remaining in the nucleus. The dual genetic origin and a vast difference of 2-3 orders of magnitude in genc-copy number between nucleus and plastids implies that the synthcsis and assembly of thylakoid polypeptides must be highly coordinated in time and space, with regard to stoichiometry, and in response to external stimuli.Light plays a crucial role among the various factors that influence the biosynthesis of the photosynthetic membranc and appears to operate at various levels. The light-dependent synthesis of polypeptides made in plastids is generally controlled post-transcriptionally (Herrmann et al

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Isolation of cDNA clones for fourteen nuclear-encoded thylakoid membrane proteins

Cited by 95 publications

References 31 publications

Purification and membrane topology of PSI‐D and PSI‐E, two subunits of the photosystem I reaction center

Purification and membrane topology of PSI‐D and PSI‐E, two subunits of the photosystem I reaction center

The 64 kDa polypeptide of spinach may not be the LHCII kinase, but a lumen‐located polyphenol oxidase

Post‐transcriptional and post‐translational regulatory steps are crucial in controlling the appearance and stability of thylakoid polypeptides during the transition of etiolated tobacco seedlings to white light

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