Abstract
Mutant LF-1 of the green alga Scenedesmus obliquus has been described by Metz and co-workers (Metz, J. G., Pakrasi, H., Seibert, M., and Arntzen, C. J. (1986) FEBS Lett. 205, 269-274) to be inactive for light-driven oxygen evolution, despite a functional Photo-system II reaction center. A polypeptide, D1, implicated in the ligation of the primary photoreactants of photosystem II, was shown to migrate with an apparent higher molecular mass on LDS-PAGE in the mutant than in the wild-type (WT) strain. We show here that polypeptide D1 is synthesized in a precursor form in Scenedesmus WT. Following synthesis and insertion into the thylakoid membrane, a 1.5-2-kDa oligopeptide is clipped off with a half-time of 1-2 min, yielding the mature 34-kDa form of the polypeptide. No processing of polypeptide D1 from mutant LF-1 was observed to take place. We show here that polypeptide D1 of LF-1 displays an identical proteolytic fingerprint pattern to the precursor D1 polypeptide of the wild-type strain. These both have molecular masses about 1.5-2 kDa higher than that of the mature WT polypeptide. A polyclonal antibody elicited by a synthetic oligopeptide (14-mer), predicted from the psbA gene nucleotide sequence to be homologous to the COOH terminus of the precursor D1 of spinach, cross-reacts only with D1 of mutant LF-1 and not with mature D1 of spinach, Chlamydomonas, or of Scenedesmus WT. This observation demonstrates that the greater molecular mass of polypeptide D1 from mutant LF-1 and of Scenedesmus WT precursor D1 is derived from a COOH-terminal extension. We conclude that the LF-1 mutant lacks the appropriate nuclear-encoded protease which processes polypeptide D1 at its COOH terminus from the precursor to the mature form. Such processing would appear to be a necessary step toward the stable incorporation of manganese into the oxygen-evolving site.
Highlights
Mutant LF- 1of the green algSacenedesmus obliquus evolution
Aside from the contamination by PSI reaction center polypeptides in the higher yield LF-1 preparation (CPI 120 kDa and minor polypeptides 15-25 kDa), the major difference between the WT and LF-c1ore preparations lies in a diffuse band located between 34 and 36 kDa which migrates with an be synthesized in a precursor form which becomes processed with increasing concentrations of proteases
This was done to yield the mature form [10,11,12]. We looked for such proc- by isolating D l on Li dodecyl sulfate (LDS)-PAGE from WTandLF-1 core essingin the WT anLdF-1 strains of Scenedesmusby carrying preparations and thenpartially proteolyzingthis polypeptide, out a pulse-chase experiment involving short labeling times according to Cleveland et al [23], using papain and S. aureus followedby rapid breakage of the cells
Summary
Typical Photosystem I1 core preparations for Scenedesmus WT and LF-1 acroempared to thatfor Chlamydomonas (Fig. 1, fourth set of lanes). This was done to yield the mature form [10,11,12] We looked for such proc- by isolating D l on LDS-PAGE from WTandLF-1 core essingin the WT anLdF-1 strains of Scenedesmusby carrying preparations and thenpartially proteolyzingthis polypeptide, out a pulse-chase experiment involving short labeling times according to Cleveland et al [23], using papain and S. aureus followedby rapid breakage of the cells. Such a pulse-chase V8 protease (Fig. 3, A and B ).
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