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

作     者：Corinne Yoshihara Kentaro Inoue Denise Schichnes Steven Ruzin William Inwood Sydney Kustu 

作者机构：Department of Plant and Microbial Biology 111 Koshland Hall University of California Berkeley CA 94720-3102 USA Department of Plant Sciences 131 Asmundson Hall University of California One Shields Avenue Davis CA 95616 USA CNR Biological Imaging Facility 381 Koshland Hall University of California Berkeley CA 94720-3102 USA 

出 版 物：《Molecular Plant》 (分子植物（英文版）)

年 卷 期：2008年第1卷第6期

页      面：1007-1020页

核心收录：

学科分类：0710[理学-生物学] 071001[理学-植物学] 07[理学] 

基　　金：美国国立卫生研究院(NIH)项目 the Torrey Mesa Research Institute, Syngenta Research Technology, La Jolla, California (S.K.) 

主　　题：CO2 acquisition fluorescence imaging membrane biology protein targeting. 

摘      要：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.