The SM protein VPS-45 is required for RAB-5-dependent endocytic transport in Caenorhabditis elegans

Abstract

Rab5, a small guanosine triphosphatase, is known to regulate the tethering and docking reaction leading to SNARE (soluble NSF attachment protein receptors)-mediated fusion between endosomes. However, it is uncertain how the signal of the activated Rab5 protein is transduced by its downstream effectors during endosome fusion. Here, we show that the Sec1/Munc18 gene vps-45 is essential for not only viability and development but also receptor-mediated and fluid-phase endocytosis pathways in Caenorhabditis elegans. We found that VPS-45 interacts with a Rab5 effector, Rabenosyn-5 (RABS-5), and the mutants of both vps-45 and rabs-5 show similar endocytic phenotypes. In the macrophage-like cells of vps-45 and rabs-5 mutants, aberrantly small endosomes were accumulated, and the endosome fusion stimulated by the mutant RAB-5 (Q78L) is suppressed by these mutations. Our results indicate that VPS-45 is a key molecule that functions downstream from RAB-5, cooperating with RABS-5, to regulate the dynamics of the endocytic system in multicellular organisms.

Figure 1

Endocytosis defects in the vps-45 mutant. (A) Receptor-mediated endocytosis-defective (Rme) phenotype. Adult hermaphrodites expressing the YP170/EGFP are shown. Arrowheads indicate oocytes endocytosing the fusion protein. Asterisks show the nucleus of oocytes. SP, spermatheca. Scale bar, 20 μm. (B) Coelomocyte uptake-defective (Cup) phenotype. Adult hermaphrodites carrying Pmyo-3∷ssGFP secrete GFP from their muscles into the body cavity. The upper panels show worms at a low magnification and the lower panels show individual coelomocytes at a high magnification. Arrowheads indicate coelomocytes endocytosing GFP. Arrows indicate pseudocoelomic GFP. Individual coelomocytes are outlined in white. Scale bars, 50 μm (upper) and 5 μm (lower). DIC, differential interference contrast; GFP, green fluorescent protein (EGFP, enhanced GFP).

Figure 2

Impairments of Texas-Red-conjugated BSA uptake by vps-45 mutant coelomocytes. The confocal micrographs show a typical pattern of TR-BSA fluorescence (red) endocytosed by coelomocytes. After microinjection of TR-BSA into the pseudocoelom, animals expressing RME-8/GFP for an endosomal marker were incubated for the indicated time (left). The upper panels show WT and the lower panels show the vps-45 mutant. Arrowheads indicate concentrations of TR-BSA in compartments labelled with RME-8/GFP. Arrows indicate RME-8/GFP-negative vesicles, probably lysosomes (see supplementary Fig 3 online). Scale bar: 5 μm in all images. Rme, receptor-mediated endocytosis defective; TR–BSA, Texas-Red-conjugated BSA.

Figure 3

C. elegans Rabenosyn-5 mutant shows a phenocopy of the vps-45 mutant in various endocytic defects. (A) Yeast two-hybrid assay. (B) Receptor-mediated endocytosis-defective (Rme) and coelomocyte uptake (Cup) phenotypes in Rabenosyn-5 (rabs-5) mutant. (C) Individual coelomocytes from animals expressing RME-8/GFP. (D) Quantitative analysis of (C). Error bars indicate s.e.m. ^*P<0.001. Scale bars, 20 μm (B, upper), 50 μm (B, lower) and 5 μm (C). DIC, differential interference contrast; GFP, green fluorescent protein.

Figure 4

vps-45 and rabs-5 mutations suppress RAB-5 (Q78L)-induced endosome fusion. (A) Individual coelomocytes from wild-type (WT) or vps-45, carrying the Pmyo-3∷ssGFP array and RAB-5 (Q78L) array (+RAB-5Q78L), or carrying only the Pmyo-3∷ssGFP array (−RAB-5 (Q78L)). RAB-5 (Q78L)-induced large endosomes accumulating GFP were detectable in WT (arrow) but not in the vps-45 mutant background. Scale bar, 5 μm. (B) Quantitative analysis of (A). In coelomocytes with red fluorescence, percentages of the coelomocyte with large endosomes per total coelomocyte were calculated. Error bars indicate s.e.m. ^*P<0.001. GFP, green fluorescent protein; RAB-5, Rab5 homologue; RABS-5, Rabenosyn-5; SYN-13 and SYN-16, Syntaxin; WT, wild type.