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Role of the EF-hand and coiled-coil domains of human Rab44 in localisation and organelle formation

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 overexpr...

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Autores principales: Ogawa, Kohei, Kadowaki, Tomoko, Tokuhisa, Mitsuko, Yamaguchi, Yu, Umeda, Masahiro, Tsukuba, Takayuki
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7645795/
https://www.ncbi.nlm.nih.gov/pubmed/33154405
http://dx.doi.org/10.1038/s41598-020-75897-7
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author Ogawa, Kohei
Kadowaki, Tomoko
Tokuhisa, Mitsuko
Yamaguchi, Yu
Umeda, Masahiro
Tsukuba, Takayuki
author_facet Ogawa, Kohei
Kadowaki, Tomoko
Tokuhisa, Mitsuko
Yamaguchi, Yu
Umeda, Masahiro
Tsukuba, Takayuki
author_sort Ogawa, Kohei
collection PubMed
description 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.
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spelling pubmed-76457952020-11-06 Role of the EF-hand and coiled-coil domains of human Rab44 in localisation and organelle formation Ogawa, Kohei Kadowaki, Tomoko Tokuhisa, Mitsuko Yamaguchi, Yu Umeda, Masahiro Tsukuba, Takayuki Sci Rep Article 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. Nature Publishing Group UK 2020-11-05 /pmc/articles/PMC7645795/ /pubmed/33154405 http://dx.doi.org/10.1038/s41598-020-75897-7 Text en © The Author(s) 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
spellingShingle Article
Ogawa, Kohei
Kadowaki, Tomoko
Tokuhisa, Mitsuko
Yamaguchi, Yu
Umeda, Masahiro
Tsukuba, Takayuki
Role of the EF-hand and coiled-coil domains of human Rab44 in localisation and organelle formation
title Role of the EF-hand and coiled-coil domains of human Rab44 in localisation and organelle formation
title_full Role of the EF-hand and coiled-coil domains of human Rab44 in localisation and organelle formation
title_fullStr Role of the EF-hand and coiled-coil domains of human Rab44 in localisation and organelle formation
title_full_unstemmed Role of the EF-hand and coiled-coil domains of human Rab44 in localisation and organelle formation
title_short Role of the EF-hand and coiled-coil domains of human Rab44 in localisation and organelle formation
title_sort role of the ef-hand and coiled-coil domains of human rab44 in localisation and organelle formation
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7645795/
https://www.ncbi.nlm.nih.gov/pubmed/33154405
http://dx.doi.org/10.1038/s41598-020-75897-7
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