Wu Q, Sandrock T M, Turgeon B G, Yoder O C, Wirsel S G, Aist J R
Department of Plant Pathology, Cornell University, Ithaca, New York 14853, USA.
Mol Biol Cell. 1998 Jan;9(1):89-101. doi: 10.1091/mbc.9.1.89.
A gene (NhKIN1) encoding a kinesin was cloned from Nectria haematococca genomic DNA by polymerase chain reaction amplification, using primers corresponding to conserved regions of known kinesin-encoding genes. Sequence analysis showed that NhKIN1 belongs to the subfamily of conventional kinesins and is distinct from any of the currently designated kinesin-related protein subfamilies. Deletion of NhKIN1 by transformation-mediated homologous recombination caused several dramatic phenotypes: a 50% reduction in colony growth rate, helical or wavy hyphae with reduced diameter, and subcellular abnormalities including withdrawal of mitochondria from the growing hyphal apex and reduction in the size of the Spitzenkörper, an apical aggregate of secretory vesicles. The effects on mitochondria and Spitzenkörper were not due to altered microtubule distribution, as microtubules were abundant throughout the length of hyphal tip cells of the mutant. The rate of spindle elongation during anaphase B of mitosis was reduced 11%, but the rate was not significantly different from that of wild type. This lack of a substantial mitotic phenotype is consistent with the primary role of the conventional kinesins in organelle motility rather than mitosis. Our results provide further evidence that the microtubule-based motility mechanism has a direct role in apical transport of secretory vesicles and the first evidence for its role in apical transport of mitochondria in a filamentous fungus. They also include a unique demonstration that a microtubule-based motor protein is essential for normal positioning of the Spitzenkörper, thus providing a new insight into the cellular basis for the aberrant hyphal morphology.
通过聚合酶链反应扩增，使用与已知驱动蛋白编码基因保守区域对应的引物，从血红色丛赤壳菌基因组DNA中克隆出一个编码驱动蛋白的基因（NhKIN1）。序列分析表明，NhKIN1属于传统驱动蛋白亚家族，与目前指定的任何驱动蛋白相关蛋白亚家族都不同。通过转化介导的同源重组缺失NhKIN1会导致几种显著的表型：菌落生长速率降低50%，菌丝呈螺旋状或波浪状且直径减小，以及亚细胞异常，包括线粒体从生长的菌丝顶端退缩和分泌小泡顶端聚集体即Spitzenkörper的大小减小。对线粒体和Spitzenkörper的影响并非由于微管分布改变，因为在突变体的菌丝顶端细胞全长中微管都很丰富。有丝分裂后期B纺锤体伸长速率降低了11%，但该速率与野生型无显著差异。这种缺乏明显有丝分裂表型的情况与传统驱动蛋白在细胞器运动而非有丝分裂中的主要作用一致。我们的结果进一步证明基于微管的运动机制在分泌小泡的顶端运输中起直接作用，并且首次证明其在丝状真菌中线粒体顶端运输中的作用。它们还独特地证明了基于微管的运动蛋白对于Spitzenkörper的正常定位至关重要，从而为异常菌丝形态的细胞基础提供了新的见解。
