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

Kohei Ogawa,Tomoko Kadowaki,Mitsuko Tokuhisa,Masahiro Umeda,Takayuki Tsukuba,Yu Yamaguchi

doi:10.1038/s41598-020-75897-7

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 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.

Highlights

Rab[44] is a large Rab GTPase that contains an amino-terminal EF-hand domain, a coiled-coil domain, and a carboxyl-terminal Rab GTPase domain
Rab GTPases translocate between the cytosol and membranes by undergoing conformational changes that are strictly controlled by two important regulators: the guanine nucleotide exchange factor (GEF) and GTPase activating protein (GAP)[5,6]
We investigated the mechanisms involved in the localisation of human Rab[44] by ectopically expressing various Rab[44] mutants in HeLa cells, which are human epithelial cells most often used in intracellular localisation analyses

Summary

Introduction

Rab[44] is a large Rab GTPase that contains an amino-terminal EF-hand domain, a coiled-coil domain, and a carboxyl-terminal Rab GTPase domain. The EF- hand domain is likely required for the partial translocation of Rab[44] to the plasma membrane and cytosol following transient Ca2+ influx, and the coiled-coil domain appears to be important for localisation and organelle formation. Comparing the sequence of the human and mouse Rab[44] reveals that both Rab[44] proteins contain EF-hand, coiled-coil, and Rab-GTPase domains. Our recent studies reported that mouse mast cells and bone-marrow cells have two isoforms: the long form including the EF-hand domain and the short form lacking this d omain[18,19]. We investigated the mechanisms involved in the localisation of human Rab[44] by ectopically expressing various Rab[44] mutants in HeLa cells, which are human epithelial cells most often used in intracellular localisation analyses

Methods

Results

Conclusion