6533b827fe1ef96bd128707e

RESEARCH PRODUCT

Expression of a higher plant light-harvesting chlorophyll a/b-binding protein in Synechocystis sp. PCC 6803

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A chimeric lhcb gene, coding for Lhcb, a higher plant chlorophyll a/b-binding light-harvesting complex of photosystem II (LHCII), was constructed using the Synechocystis sp. PCC 6803 psbA3 promoter and a modified lhcb gene from pea. This construct drives synthesis of full-length, mature Lhcb under the control of the strong psbA3 promoter that usually drives expression of the D1 protein of photosystem II. This chimeric gene was transformed into a photosystem I-less/chlL(-) Synechocystis sp. PCC 6803 strain that is unable to synthesize chlorophyll in darkness. In the resulting strain, a high level of lhcb transcript was detected and transcript accumulation was enhanced by addition of exogenous Zn-chlorophyllide b. The chimeric lhcb gene was translated to produce full-length Lhcb as demonstrated by pulse-labeling: a new radioactively labeled band of a size corresponding to full-length Lhcb was visible on autoradiograms. Using Triton X-114 phase fractionation, this labeled protein band was found to partition to the phase containing integral membrane proteins, indicating that the pulse-labeled Lhcb is readily integrated into the membrane. However, Lhcb was rapidly degraded and did not accumulate in thylakoid membranes to levels that were detectable other than by pulse labeling. Upon immunological detection with LHCII antibodies, a small protein (approximately 8 kDa) was found specifically in the lhcb-containing mutant. We interpret this protein to be a degradation product of the full-length Lhcb. This fragment was stabilized by supplementing cells with xanthophylls, which incorporated into thylakoid membranes only in the mutant carrying lhcb. The lutein/chlorophyll ratio of thylakoids of this mutant was about 1 : 10. These results indicate that in this cyanobacterial system Lhcb is synthesized, integrated into the membrane, and then degraded to a approximately 8 kDa fragment that is stabilized by pigment binding and does not require the presence of chlorophyll b.