The Chlamydomonas FLA10 gene encodes a novel kinesin-homologous protein.
Many genes on the uni linkage group of Chlamydomonas affect the basal body/flagellar apparatus. Among these are five FLA genes, whose mutations cause temperature-sensitive defects in flagellar assembly. We present the molecular analysis of a gene which maps to fla10 and functionally rescues the fla10 phenotype. Nucleotide sequencing revealed that the gene encodes a kinesin-homologous protein, KHP1. The 87-kD predicted KHP1 protein, like kinesin heavy chain, has an amino-terminal motor domain, a central alpha-helical stalk, and a basic, globular carboxy-terminal tail. Comparison to other kinesin superfamily members indicated striking similarity (64% identity in motor domains) to a mouse gene, KIF3, expressed primarily in cerebellum. In synchronized cultures, the KHP1 mRNA accumulated after cell division, as did flagellar dynein mRNAs. KHP1 mRNA levels also increased following deflagellation. Polyclonal antibodies detected KHP1 protein in Western blots of purified flagella and axonemes. The protein was partially released from axonemes with ATP treatment, but not with AMP-PNP. Western blot analysis of axonemes from various motility mutants suggested that KHP1 is not a component of radial spokes, dynein arms, or the central pair complex. The quantity of KHP1 protein in axonemes of the mutant fla10-1 was markedly reduced, although no reduction was observed in two other uni linkage group mutants, fla9 and fla11. Furthermore, fla10-1 was rescued by transformation with KHP1 genomic DNA. These results indicate that KHP1 is the gene product of FLA10 and suggest a novel role for this kinesin-related protein in flagellar assembly and maintenance.
Rockefeller University, New York 10021.
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Walther
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Vashishtha
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JL
Hall
D020418:Algal Proteins; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002453:Cell Cycle / Q000235:genetics; D016825:Chlamydomonas reinhardtii / Q000235:genetics*; D003001:Cloning, Molecular; D005407:Flagella / Q000737:chemistry* / Q000648:ultrastructure; D017125:Genes, Protozoan / Q000235:genetics*; D016547:Kinesins / Q000235:genetics; D008869:Microtubule-Associated Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D009024:Morphogenesis; D015800:Protozoan Proteins / Q000235:genetics / Q000302:isolation & purification; D012333:RNA, Messenger / Q000032:analysis; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D020418:Algal Proteins; C088230:KHP1 protein, Chlamydomonas; D008869:Microtubule-Associated Proteins; D015800:Protozoan Proteins; D012333:RNA, Messenger; D016547:Kinesins