<i>In Vitro</i> Processing of Precursors of Thylakoid Membrane Proteins of <i>Chlamydomonas reinhardtii</i> y-1

Marks, Dawn B.; Keller, Barbara; Hoober, J. Kenneth

doi:10.1104/pp.79.1.108

Cited by 16 publications

(16 citation statements)

References 25 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…These results imply that either processing is coupled to integration or that only the membrane-integrated pLHCP is substrate for the processing protease. This latter possibility is consistent with the interpretations of Marks et al (18) (Fig. 4a, M; Fig.…”

Section: Methodssupporting

confidence: 93%

Light-Harvesting Chlorophyll a/b Protein

Cline¹

1988

Plant Physiol.

View full text Add to dashboard Cite

The apoprotein of the light-harvesting chlorophyll alb protein (LHCP) is a major integral thylakoid membrane protein that is normally complexed with chlorophyll and xanthophylls and serves as the antenna complex of photosystem IL LHCP is encoded in the nucleus and synthesized in the cytosol as a higher molecular weight precursor that is subsequently imported into chloroplasts and assembled into thylakoids. In a previous study it was established that the LHCP precursor can integrate into isolated thylakoid membranes. The present study demonstrates that under conditions designed to preserve thylakoid structure, the inserted LHCP precursor is processed to mature size, assembled into the LHC II chlorophyll-protein complex, and localized to the appressed thylakoid membranes. Under these conditions, light can partially replace exogenous ATP in the membrane integration process.Thylakoid membranes contain approximately 45 different polypeptides, most of which are organized into four distinct supramolecular complexes: PSI, PSII, Cyt b/f, and the ATP synthase (11,23). In most plant chloroplasts, these complexes are nonuniformly distributed into two different structural thylakoid domains, the appressed membranes and the nonappressed membranes. PSII complexes are located predominantly in the appressed membranes, PSI and ATP synthase are located exclusively in the nonappressed membranes, and the Cyt blf complex appears to be present in both domains (23).Biogenesis of thylakoid protein complexes is an intriguing but little understood process. Each complex contains nuclearly encoded as well as chloroplast-encoded polypeptides (11). Thus, complex formation involves coordinate assembly of polypeptides that derive from two different cellular compartments. Chloroplast-encoded polypeptides are synthesized at or near their functional locations on thylakoid-bound ribosomes (21). Conversely, nuclearly encoded polypeptides must be imported from their site of synthesis in the cytosol (29). The fact that thylakoids are spatially separated from the delimiting envelope membranes indicates that assembly of these polypeptides is a multistep process, involving interaction with three different membranes. The assembly pathway for cytosolically synthesized thylakoid proteins has not been definitively established. However, recent studies (9, 30) suggest that these proteins are translocated across the envelope into the aqueous stromal matrix, where they are assembled into thylakoids by a second protein translocation/integration process.

show abstract

Section: Methodssupporting

confidence: 93%

Light-Harvesting Chlorophyll a/b Protein

Cline¹

1988

Plant Physiol.

View full text Add to dashboard Cite

show abstract

“…We began a study of the proteases that potentially contribute to degradation of these proteins. The soluble fraction of C reinhardtii y-l contains a number of proteolytic activities (18,22). Among these is a specific protease derived from the chloroplast stroma that removes the transit sequence from precursors of the Cab proteins (22).…”

Section: Discussionmentioning

confidence: 99%

Purification and Characterization of a Membrane-Bound Protease from Chlamydomonas reinhardtii

Hoober¹,

Hughes²

1992

Plant Physiol.

Self Cite

View full text Add to dashboard Cite

In Chiamydomonas reinhardtii y-1, newly synthesized chlorophyll a/b-binding apoproteins are degraded when chlorophylls are not present for assembly of stable light-harvesting complexes. A protease was purified from the membrane fraction of degreened y-1 cells, which digested chlorophyll a/b-binding proteins in membranes from C. reinhardtii pg-1 13, a protease-deficient strain. This protease was active with p-nitroanilides of nonpolar amino acids (Leu and Phe), but not of basic amino acids (Lys and Arg). The apparent molecular weight of the enzyme is 38,000 ± 2,000 as determined by electrophoresis in the presence of sodium dodecyl sulfate. Typical inhibitors of the major classes of proteases were ineffective with this enzyme. Protease activity was constant from pH 7.5 to 9; a plot of log V versus pH suggested that deprotonation of an ionizable group with a pK value of 6.0 to 6.5 is required for activity. The protease was inactivated by diethylpyrocarbonate and by photooxidation sensitized by rose bengal. These results suggested that a histidyl residue is required for catalysis. Although very sensitive to photodynamic conditions in vitro, the enzyme was not inactivated in vivo when cells were exposed to light.

show abstract

“…Immunoprecipitation. As previously described (2,5) Electrophoresis. Electrophoresis on 10 to 20% polyacrylamide gradient gels, autoradiography, scanning, and determination of radioactivity in protein peaks were performed as previously described (2,5).…”

Section: Methodsmentioning

confidence: 99%

“…Assignment of precursor-product relationships between the two LHCP precursors and their three structurally similar, mature products in Chlamydomonas reinhardtii y-l was facilitated by our observation that when the precursors were bound to antibodies, only one of the two was cleaved to its products (5). We now report that binding of the RbcS precursor (pRbcS) to antibodies inhibited cleavage to its mature product and permitted observation of a secondary processing site.…”

mentioning

confidence: 94%