• BMB Reports
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• 제40권1호
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• pp.44-52
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• 2007

Kinesins, as a kind of microtubule-based motor proteins, have a conserved microtubule-binding site in their motor domain. Here we report that two homologous kinesins in Arabidopsis thaliana, KatB and KatC, contain a second microtubule-binding site in their tail domains. The prokaryotic-expressed N-terminal tail domain of the KatC heavy chain can bind to microtubules in an ATP-insensitive manner. To identify the precise region responsible for the binding, a serious of truncated KatC cDNAs encoding KatC N-terminal regions in different lengths, KatC1-128, KatC1-86, KatC1-73 and KatC1-63, fused to Histidine-tags, were expressed in E. coli and affinity-purified. Microtubule cosedimentation assays show that the site at amino acid residues 74-86 in KatC is important for microtubule-binding. By similarity, we obtained three different lengths of KatB N-terminal regions, KatB1-384, KatB1-77, and KatB1-63, and analyzed their microtubule-binding ability. Cosedimentation assays indicate that the KatB tail domain can also bind to microtubules at the same site as and in a similar manner to KatC. Fluorescence microscopic observations show that the microtubule-binding site at the tail domain of KatB or KatC can induce microtubules bundling only when the stalk domain is present. Through pull-down assays, we show that KatB1-385 and KatC1-394 are able to interact specifically with themselves and with each other in vitro. These findings are significant for identifying a previously uncharacterized microtubule-binding site in the two kinesin proteins, KatB and KatC, and the functional relations between them.

• BMB Reports
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• 제40권5호
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• pp.723-730
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• 2007

Kinesin is an ATP-driven microtubule motor protein that plays important roles in control of microtubule dynamics, intracellular transport, cell division and signal transduction. The kinesin superfamily is composed of numerous members that are classified into 14 subfamilies. Animal kinesins have been well characterized. In contrast, plant kinesins have not yet to be characterized adequately. Here, a novel plant-specific kinesin gene, GhKCH2, has been cloned from cotton (Gossypium hirsutum) fibers and biochemically identified by prokaryotic expression, affinity purification, ATPase activity assay and microtubule-binding analysis. The putative motor domain of GhKCH2, $M_{396-734}$ corresponding to amino acids Q396-N734 was fused with 6$\times$His-tag, soluble-expressed in E. coli and affinity-purified in a large amount. The biochemical analysis demonstrated that the basal ATPase activity of $M_{396-734}$ is not activated by $Ca^{2+}$, but stimulated 30-fold max by microtubules. The enzymatic activation is microtubule-concentration-dependent, and the concentration of microtubules that corresponds to half-maximum activation was about 11 ${\mu}M$, much higher than that of other kinesins reported. The cosedimentation assay indicated that $M_{396-734}$ could bind to microtubules in vitro whenever the nucleotide AMP-PNP is present or absent. As a plant-specific microtubule-dependent kinesin with a lower microtubule-affinity and a nucleotide-independent microtubule-binding ability, cotton GhKCH2 might be involved in the function of microtubules during the deposition of cellulose microfibrils in fibers or the formation of cell wall.

• 생명과학회지
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• 제17권2호통권82호
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• pp.204-210
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• 2007

평활근 ${\alpha}$-트로포마이오신의 높은 액틴 친화력은 아미노산 잔기 Gln276 및 Thr277에 기인한다는 이전 보고에 따라, 2 잔기 중 어느 잔기가 액틴 친화력에 더 중요한 가를 알아보기 위하여 골격근 트로포마이오신의 His 혹은 Ala 단일 잔기를 각각 Gln 혹은 Thr으로 치환한 돌연변이 트로포마이오신을 제작하여 대장균에서 대량발현 시킨 후 정제하여 액틴 결합력을 측정하였다. 비록 비아세틸화된 트로포마이오신의 경우 Gln 및 Thr 잔기가 최고 액틴친화력을 위해 모두 필요하나, 돌연변이 트로포마이신 중 Gln 잔기를 가진 돌연변이 트로포마이오신들이 다른 돌연변이 트로포마이오신들에 비하여 3에서 4배 높은 액틴친화력을 보였다. 이러한 결과는 평활근 ${\alpha}$-트로포마이오신의 높은 액틴 친화력은 Thr277 잔기보다 Gln276 잔기에 주로 기인한다는 것을 의미한다.