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标题：Role of the EF-hand and coiled-coil domains of human Rab44 in localisation and organelle formation
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作者：Kohei Ogawa ; Tomoko Kadowaki ; Mitsuko Tokuhisa 等
期刊名称：Scientific Reports
电子版ISSN：2045-2322
出版年度：2020
卷号：10
期号：1
页码：1-23
DOI：10.1038/s41598-020-75897-7
出版社：Springer Nature
摘要：Rab44 is a large Rab GTPase that contains an amino-terminal EF-hand domain, a coiled-coil domain, and a carboxyl-terminal Rab GTPase domain. However, the roles of the EF-hand and coiled-coil domains remain unclear. Here, we constructed various deletion and point mutants of human Rab44. When overexpressed in HeLa cells, the wild-type Rab44 (hWT) formed ring-like structures, and partially localised to lysosomes. The dominant negative mutant, hT847N, localised to lysosomes and the cytosol, while the constitutively active mutant, hQ892L, formed ring-like structures, and partially localised to the plasma membrane and nuclei. The hΔEF, hΔcoil, and h826-1021 mutants also formed ring-like structures; however, their localisation patterns differed from hWT. Analysis of live imaging with LysoTracker revealed that the size of LysoTracker-positive vesicles was altered by all other mutations than the hC1019A and hΔEF. Treatment with ionomycin, a Ca2 ionophore, induced the translocation of hWT and hΔcoil into the plasma membrane and cytosol, but had no effect on the localisation of the hΔEF and h826-1021 mutants. Thus, the EF- hand domain is likely required for the partial translocation of Rab44 to the plasma membrane and cytosol following transient Ca2 influx, and the coiled-coil domain appears to be important for localisation and organelle formation.
其他摘要：Abstract Rab44 is a large Rab GTPase that contains an amino-terminal EF-hand domain, a coiled-coil domain, and a carboxyl-terminal Rab GTPase domain. However, the roles of the EF-hand and coiled-coil domains remain unclear. Here, we constructed various deletion and point mutants of human Rab44. When overexpressed in HeLa cells, the wild-type Rab44 (hWT) formed ring-like structures, and partially localised to lysosomes. The dominant negative mutant, hT847N, localised to lysosomes and the cytosol, while the constitutively active mutant, hQ892L, formed ring-like structures, and partially localised to the plasma membrane and nuclei. The hΔEF, hΔcoil, and h826-1021 mutants also formed ring-like structures; however, their localisation patterns differed from hWT. Analysis of live imaging with LysoTracker revealed that the size of LysoTracker-positive vesicles was altered by all other mutations than the hC1019A and hΔEF. Treatment with ionomycin, a Ca 2 ionophore, induced the translocation of hWT and hΔcoil into the plasma membrane and cytosol, but had no effect on the localisation of the hΔEF and h826-1021 mutants. Thus, the EF- hand domain is likely required for the partial translocation of Rab44 to the plasma membrane and cytosol following transient Ca 2 influx, and the coiled-coil domain appears to be important for localisation and organelle formation.