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

Sci Rep. 2020 Nov 5;10(1):19149. 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Membrane / metabolism*
  • Cell Nucleus / metabolism*
  • EF Hand Motifs / physiology*
  • HeLa Cells
  • Humans
  • Organelle Shape / physiology
  • Protein Domains
  • rab GTP-Binding Proteins / metabolism*

Substances

  • rab GTP-Binding Proteins