The major Rhesus （Rh） protein of the green alga Chlamydomonas reinhardtii, Rhl, is homologous to Rh proteins of humans. It is an integral membrane protein involved in transport of carbon dioxide. To localize a fusio...The major Rhesus （Rh） protein of the green alga Chlamydomonas reinhardtii, Rhl, is homologous to Rh proteins of humans. It is an integral membrane protein involved in transport of carbon dioxide. To localize a fusion of intact Rhl to the green fluorescent protein （GFP）, we used as host a white （Its1） mutant strain of C. reinhardtii, which is blocked at the first step of carotenoid biosynthesis. The Its1 mutant strain accumulated normal amounts of Rhl heterotrophically in the dark and Rhl-GFP was at the periphery of the cell co-localized with the cytoplasmic membrane dye FM4-64. Although Rhl carries a potential chloroplast targeting sequence at its N-terminus, Rhl-GFP was clearly not associated with the chloroplast envelope membrane. Moreover, the N-terminal half of the protein was not imported into chloroplasts in vitro and N-terminal regions of Rhl did not direct import of the small subunit of ribulose bisphosphate carboxylase （SSU）. Despite caveats to this interpretation, which we discuss, current evidence indicates that Rhl is a cytoplasmic membrane protein and that Rhl-GFP is among the first cytoplasmic membrane protein fusions to be obtained in C. reinhardtii. Although Its1 （white） mutant strains cannot be used to localize proteins within sub-compartments of the chloroplast because they lack thylakoid membranes, they should nonetheless be valuable for localizing many GFP fusions in Chlamydomonas.展开更多
文摘The major Rhesus （Rh） protein of the green alga Chlamydomonas reinhardtii, Rhl, is homologous to Rh proteins of humans. It is an integral membrane protein involved in transport of carbon dioxide. To localize a fusion of intact Rhl to the green fluorescent protein （GFP）, we used as host a white （Its1） mutant strain of C. reinhardtii, which is blocked at the first step of carotenoid biosynthesis. The Its1 mutant strain accumulated normal amounts of Rhl heterotrophically in the dark and Rhl-GFP was at the periphery of the cell co-localized with the cytoplasmic membrane dye FM4-64. Although Rhl carries a potential chloroplast targeting sequence at its N-terminus, Rhl-GFP was clearly not associated with the chloroplast envelope membrane. Moreover, the N-terminal half of the protein was not imported into chloroplasts in vitro and N-terminal regions of Rhl did not direct import of the small subunit of ribulose bisphosphate carboxylase （SSU）. Despite caveats to this interpretation, which we discuss, current evidence indicates that Rhl is a cytoplasmic membrane protein and that Rhl-GFP is among the first cytoplasmic membrane protein fusions to be obtained in C. reinhardtii. Although Its1 （white） mutant strains cannot be used to localize proteins within sub-compartments of the chloroplast because they lack thylakoid membranes, they should nonetheless be valuable for localizing many GFP fusions in Chlamydomonas.
