Acidic clusters target transmembrane proteins to the contractile vacuole inDictyosteliumcells

Abstract

The mechanisms responsible for the targeting of transmembrane integral proteins to the contractile vacuole (CV) network in Dictyostelium discoideum are unknown. Here we show that the transfer of the cytoplasmic domain of a CV-resident protein (Rh50) to a reporter transmembrane protein (CsA) is sufficient to address the chimera (CsA-Rh50) to the CV. We identified two clusters of acidic residues responsible for this targeting, and these motifs interacted with the gamma-adaptin AP-1 subunit in a yeast protein-protein interaction assay. For the first time we report the existence of an indirect transport pathway from the plasma membrane to the CV via endosomes. Upon internalization, the small fraction of CsA-Rh50 present at the cell surface was first concentrated in endosomes distinct from early and late p80-positive endosomes and then slowly transported to the CV. Together our results suggest the existence of an AP-1-dependent selective transport to the contractile vacuole in Dictyostelium

Dynamics of clathrin-mediated endocytosis and its requirement for organelle biogenesis in Dictyostelium

“…This is in agreement with results for Rh50 showing that a fusion protein with the cytoplasmic tail of Rh50 traffics via the plasma membrane en route to the contractile vacuole (Mercanti et al, 2006a). It also suggests the plasma membrane is a potential source of membrane for the contractile vacuole.…”

The inverse BAR-domain protein IBARa drives membrane remodelling to control osmoregulation, phagocytosis and cytokinesis

“…As a consequence, the opposite side of IBARa-N is oriented towards the cytosol, with the negatively charged patch in the middle of the IBARa-N concave side being accessible for potential protein-protein interactions. As previously reported for other proteins (Mercanti et al, 2006), the acidic patch might also serve as a localization signal to the CV.…”

“…S8) -presumably the contractile vacuole (CV), which is an osmoregulatory organelle (Gabriel et al, 1999). To confirm this, fixed cells were additionally stained for endosomes, as well as for the CV marker csA-Rh50, after ectopic expression of this construct in wild-type (WT) cells (Mercanti et al, 2006). As shown in Fig.…”

“…All constructs were verified by sequencing. The plasmids for expression of GFP-Dajumin, GFPmyosin-VII and csA-Rho50 were as described previously (Gabriel et al, 1999;Tuxworth et al, 2001;Mercanti et al, 2006).…”

The Rh protein family: gene evolution, membrane biology, and disease association

“…The only exception is RhgA from D. discoideum, which resides in contractile vacuoles [126], organelles that play a key role in osmoregulation [127]. A stretch of negatively charged residues clustered in the C-tail of RhgA appears crucial for this targeting [128]. In human red cells, Rh30 and RhAG are vertically linked to the cytoskeleton through their C-tails.…”