Mutations in the SUP-PF-1 locus of Chlamydomonas reinhardtii identify a regulatory domain in the beta-dynein heavy chain.
Mutations in the SUP-PF-1 locus of Chlamydomonas reinhardtii identify a regulatory domain in the beta-dynein heavy chain.
复制标题
莱茵衣藻 SUP-PF-1 基因座的突变确定了 β-动力蛋白重链中的调节域。
DOI：
10.1083/jcb.126.6.1495
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发表时间：
1994
期刊：
影响因子：
--
通讯作者：
Dutcher,SK
中科院分区：
文献类型：
--
作者：
Porter,ME;Knott,JA;Gardner,LC;Mitchell,DR;Dutcher,SK
We have characterized a group of regulatory mutations that alter the activity of the outer dynein arms. Three mutations were obtained as suppressors of the paralyzed central pair mutant pf6 (Luck, DJL, and G. Piperno. 1989. Cell Movement. pp. 49-60), whereas two others were obtained as suppressors of the central pair mutant pfl6. Recombination analysis and complementation tests indicate that all five mutations are alleles at the SUP-PF-1/ODA4 locus and that each allele can restore motility to radial spoke and central pair defective strains. Restriction fragment length polymorphism analysis with a genomic probe for the/~-dynein heavy chain (DHC) gene confirms that this locus is tightly linked to the/~-DHC gene. Although all five mutant sup-pf-1 alleles alter the activity of the outer dynein arm as assayed by measurements of flagellar motility, only two alleles have a discernable polypeptide defect by SDS-PAGE. We have used photolytic and proteolytic cleavage procedures to localize the polypeptide defect to an, x, 100-kD domain downstream from the last putative nucleotide binding site. This region is encoded by~ 5 kb of genomic DNA (Mitchell, DR, and K. Brown. 1994. J. Cell Sci. 107: 653-644). PCR amplification of wild-type and mutant DNA across this region identified one PCR product that was consistently smaller in the sup-pf-1 DNA. Direct DNA sequencing of the PCR products revealed that two of the sup-pf-1 mutations are distinct, in-frame deletions. These deletions occur within a region that is predicted to encode a small oL-helical coiled-coil domain of the/~-DHC. This domain may play a role in protein-protein interactions within the outer dynein arm. Since both the size and location of this domain have been conserved in all axonemal and cytoplasmic DHCs sequenced to date, it presumably performs a common function in all dynein isoforms.T II~ dynein ATPases are a family of motor enzymes that provide the driving force for ciliary and flagellar motility and contribute to microtubule-based movements inside cells (reviewed in Porter and Johnson, 1989; Vallee, 1993). These enzymes convert the energy derived from nucleotide binding and hydrolysis into the directed movement of cellular cargoes toward the minus ends of microtubules (reviewed in Porter and Johnson, 1989). All dyneins characterized thus far are extremely large (> 1-2 MD), multisubunit protein complexes. The large size and subunit complexity have hampered the identification of functional domains within the dyneins. However, the numerous flagellar mutations that affect dynein structure and function in Chlamydomonas, coupled with the recently published sequences of dynein heavy chains (DHCs) t, provide new op-
