Abstract
The C-terminal ends of small GTPases contain hypervariable sequences which may be posttranslationally modified by defined lipid moieties. The diverse structural motifs generated direct proteins towards specific cellular membranes or organelles. However, knowledge on the factors that determine these selective associations is limited. Here we show, using advanced microscopy, that the isoprenylation and palmitoylation motif of human RhoB (–CINCCKVL) targets chimeric proteins to intraluminal vesicles of endolysosomes in human cells, displaying preferential co-localization with components of the late endocytic pathway. Moreover, this distribution is conserved in distant species, including cells from amphibians, insects and fungi. Blocking lipidic modifications results in accumulation of CINCCKVL chimeras in the cytosol, from where they can reach endolysosomes upon release of this block. Remarkably, CINCCKVL constructs are sorted to intraluminal vesicles in a cholesterol-dependent process. In the lower species, neither the C-terminal sequence of RhoB, nor the endosomal distribution of its homologs are conserved; in spite of this, CINCCKVL constructs also reach endolysosomes in Xenopus laevis and insect cells. Strikingly, this behavior is prominent in the filamentous ascomycete fungus Aspergillus nidulans, in which GFP-CINCCKVL is sorted into endosomes and vacuoles in a lipidation-dependent manner and allows monitoring endosomal movement in live fungi. In summary, the isoprenylated and palmitoylated CINCCKVL sequence constitutes a specific structure which delineates an endolysosomal sorting strategy operative in phylogenetically diverse organisms.
Highlights
Regulation of vesicular traffic is a key process for many cellular functions
Fluorescent CINCCKVL constructs localize at endolysosomal compartments in human cells To assess the ability of the CINCCKVL motif to induce endolysosomal localization in distant species, we first assessed the distribution of fluorescent CINCCKVL chimeric proteins in primary human fibroblasts, a species in which these constructs have not been characterized
We have shown that the isoprenylation and palmitoylation motif of the GTPase RhoB constitutes one such structure per se and is able to determine protein sorting to lysosomal compartments in cells from distant species, including fungi and humans
Summary
Regulation of vesicular traffic is a key process for many cellular functions. Signal transduction by membrane receptors, protein delivery to specific compartments, membrane repair and exosome shedding or autophagy, among others, depend on compartmentalized traffic of molecules within the cell. Intracellular vesicles constitute a yet not fully characterized array of dynamic membranous compartments which are in continuous evolution and transformation, resulting in the acquisition of a specific protein and lipid composition [1,2] These components can regulate the fate of proteins by sorting them through different types of intracellular vesicles that can come from or recycle back to the plasma membrane, send proteins along the secretory pathway from the Golgi or travel along trafficking routes through the late endosomal pathway to the lysosome to be degraded, among many other processes. In the case of lysosomal transmembrane proteins, specific motifs such as dileucine or tyrosine-containing sequences can interact with clathrin-associated proteins and trigger lysosomal localization [7] Modifications such as phosphorylation and acylation of proteins at particular residues can serve as vesicle-specific membrane anchors for peripheral membrane proteins [8]
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