Mital Jeffrey, Lutter Erika I, Barger Alexandra C, Dooley Cheryl A, Hackstadt Ted
Host-Parasite Interactions Section, Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840, USA; Quinnipiac University, Hamden, CT 06518, USA.
Host-Parasite Interactions Section, Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840, USA.
Biochem Biophys Res Commun. 2015 Jun 26;462(2):165-70. doi: 10.1016/j.bbrc.2015.04.116. Epub 2015 May 2.
Chlamydia trachomatis actively subverts the minus-end directed microtubule motor, dynein, to traffic along microtubule tracks to the Microtubule Organizing Center (MTOC) where it remains within a membrane bound replicative vacuole for the duration of its intracellular development. Unlike most substrates of the dynein motor, disruption of the dynactin cargo-linking complex by over-expression of the p50 dynamitin subunit does not inhibit C. trachomatis transport. A requirement for chlamydial protein synthesis to initiate this process suggests that a chlamydial product supersedes a requirement for p50 dynamitin. A yeast 2-hybrid system was used to screen the chlamydia inclusion membrane protein CT850 against a HeLa cell cDNA library and identified an interaction with the dynein light chain DYNLT1 (Tctex1). This interaction was at least partially dependent upon an (R/K-R/K-X-X-R/K) motif that is characteristic of DYNLT1 binding domains. CT850 expressed ectopically in HeLa cells localized at the MTOC and this localization is similarly dependent upon the predicted DYNLT1 binding domain. Furthermore, DYNLT1 is enriched at focal concentrations of CT850 on the chlamydial inclusion membrane that are known to interact with dynein and microtubules. Depletion of DYNLT1 disrupts the characteristic association of the inclusion membrane with centrosomes. Collectively, the results suggest that CT850 interacts with DYNLT1 to promote appropriate positioning of the inclusion at the MTOC.
沙眼衣原体能够主动破坏向负端移动的微管马达动力蛋白，使其沿着微管轨道运输至微管组织中心（MTOC），在细胞内发育期间，它会一直存在于膜结合的复制性液泡中。与动力蛋白的大多数底物不同，过表达p50动力素亚基破坏动力蛋白激活蛋白货物连接复合物，并不会抑制沙眼衣原体的运输。沙眼衣原体蛋白质合成对启动这一过程的需求表明，一种沙眼衣原体产物取代了对p50动力素的需求。利用酵母双杂交系统，针对HeLa细胞cDNA文库筛选沙眼衣原体包涵体膜蛋白CT850，并鉴定出其与动力蛋白轻链DYNLT1（Tctex1）存在相互作用。这种相互作用至少部分依赖于DYNLT1结合域特有的（R/K-R/K-X-X-R/K）基序。在HeLa细胞中异位表达的CT850定位于MTOC，这种定位同样依赖于预测的DYNLT1结合域。此外，DYNLT1在沙眼衣原体包涵体膜上CT850的局部聚集处富集，已知这些聚集处与动力蛋白和微管相互作用。DYNLT1的缺失会破坏包涵体膜与中心体的特征性结合。总体而言，结果表明CT850与DYNLT1相互作用，以促进包涵体在MTOC处的适当定位。
