An Rh1-GFP Fusion Protein Is in the Cytoplasmic Membrane of a White Mutant Strain of Chlamydomonas reinhardtii

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.

The Ultrastructure of a Chlamydomonas reinhardtii Mutant Strain Lacking Phytoene Synthase Resembles that of a Colorless Alga

Chlamydomonas reinhardtii strains lacking phytoene synthase, the first enzyme of carotenoid biosynthesis, are white. They lack carotenoid pigments, have very low levels of chlorophyll, and can grow only heterotrophically in the dark. Our electron and fluorescence microscopic studies showed that such a mutant strain （Itsl-204） had a proliferated plastid envelope membrane but no stacks of thylakoid membranes within the plastid. It accumulated cytoplasmic compartments that appeared to be autophagous vacuoles filled with membranous material. The Its1 mutants apparently lacked pyrenoid bodies, which normally house ribulose bisphosphate carboxylase-oxygenase （Rubisco）, and accumulated many starch granules. Although these mutant strains cannot synthesize the carotenoid and carotenoid-derived pigments present in the phototactic organelle （eyespot）, the mutant we examined made a vestigial eyespot that was disorganized and often mislocalized to the posterior end of the cell. The absence of a pyrenoid body, the accumulation of starch, and the disorganization of the eyespot may all result from the absence of thylakoids. The ultrastructure of Its1 mutant strains is similar to but distinct from that of previously described white and yellow mutant strains of C. reinhardtii and is similar to that of naturally colorless algae of the Polytoma group.