RME-8 physically interacts with SNX-1. (A) The interaction between RME-8 and SNX-1 requires C-terminal SNX-1 residues span 423–466. RME-8 (residues 1337–2279) was expressed in a yeast reporter strain as a fusion with the DNA-binding domain of LexA (bait). SNX-1 and its truncated forms were expressed in the same yeast cells as fusions with the B42 transcriptional activation domain (prey). Interaction between bait and prey was assayed by complementation of leucine auxotrophy (LEU2 growth assay). Colonies were diluted in liquid and spotted on solid growth medium directly or after further 10X dilution. (B) The interaction of RME-8 with SNX-1 requires RME-8 sequences C-terminal to the DNA J-domain. SNX-1 (residues 221–472) was expressed in a yeast reporter strain, a fusion with the B42 transcriptional activation domain (prey). Mutant forms of RME-8 were expressed in the same yeast cells as a fusion with the DNA-binding domain of LexA (bait). Interaction between bait and prey was assayed by complementation of leucine auxotrophy (LEU2 growth assay) as above. (C) Schematic representations of SNX-1 and RME-8 and the regions of each used in the Y2H analysis. Protein domains are displayed as boxes (white for the ARM-like domain, dark for others) above protein sequences used in the study (shown as dark lines). Amino-acid numbers are indicated. (D) Glutathione beads loaded with recombinant GST or GST-SNX-1(271–472) were incubated with in vitro expressed HA-RME-8(1337–2279), and then washed to remove unbound proteins. Bound proteins were eluted and analysed by western blot using anti-HA (top) or anti-GST (bottom) antibodies. Input lane contains in vitro expressed HA-RME-8(1337–2279) used in the binding assays (10%).

MIG-14 recycling requires RME-8 and SNX-1. Panels A-G′ show confocal micrographs of intestinally expressed MIG-14-GFP in top and middle focal planes in intact living animals. Note the reduced MIG-14-GFP intensity, and altered subcellular localization, in rme-8 and snx-1 mutants (A–H). RNAi-mediated depletion of the mu2 subunit of AP-2 (DPY-23) restored MIG-14-GFP fluorescence, and enhanced cell surface localization, in both rme-8 and snx-1 mutants (D–E′, H), arrowheads indicate lateral membrane, arrows indicate basal membrane. RNAi-mediated depletion of lysosome biogenesis protein CUP-5/mucolipin1 inhibited loss of MIG-14-GFP signal in rme-8 and snx-1 mutants (F–G′). Bar graph indicating average MIG-14-GFP fluorescence intensity calculated as described in Materials and methods (H). Asterisks indicate a significant difference in the one-tailed Student's t-test (P<0.01). P-values for MIG-14-GFP were 6.58 × 10^–7 for wild type (WT) versus rme-8(b1023) and 5.43 × 10^–7 for WT versus snx-1(tm847). Cargo proteins that recycle through the recycling endosome, the human transferrin receptor (hTfR-GFP), and the IL-2 receptor alpha chain (hTAC-GFP) were not affected in rme-8 and snx-1 mutants (I–N). For panels O–R″ images from deconvolved 3D image stacks are shown. All images were acquired in intact living animals expressing GFP and mCherry-tagged proteins specifically in intestinal epithelial cells. Autofluorescent lysosome-like organelles are shown in blue. In rme-8(b1023), snx-1(tm847), and hsp-1/hsc70(RNAi) animals, but not WT animals (O–O″), MIG-14-GFP colocalizes with late endosome and lysosome marker mCherry-RAB-7 (P–R″). MIG-14-GFP localizes to the apparent limiting membrane of the late endosomes/lysosomes as well as apparent intralumenal structures. Note that the signal intensity for MIG-14-GFP in mutant and RNAi animals was boosted to allow visualization and colocalization. Arrowheads indicate late endosomes/lysosomes labelled by both mCherry-RAB-7 and MIG-14-GFP. Insets show enlargements (× 3) of the boxed area. Scale bar represents 10 μm.

Clathrin dynamics are impaired in rme-8(b1023), snx-1(tm847), and hsp-1(RNAi) knock-down animals. (A–D″) Representative images of GFP-CHC-1 in wild-type control, rme-8(b1023), snx-1(tm847), and hsp-1(RNAi) animals before bleaching (prebleach), immediately after bleaching (bleach) and 100 s after bleaching (100 s). Bleached areas are shown by white-bordered circles. Fluorescence intensities in bleached areas were recorded every 10 s. (E) Mean time courses of FRAP (±s.d.) from eight animals of each genotype as indicated with WT (wild-type control), rme-8(b1023), snx-1(tm847), and hsp-1(RNAi). Error bars represent standard deviations from the mean (n=8 each time point, eight animals of each genotype were sampled in the intestine). Scale bar represents 10 μm.

RME-8 colocalizes with SNX-1 and RAB-5 on early endosomes. Representative images from deconvolved 3D image stacks are shown. All images were acquired in intact living animals expressing GFP and mCherry-tagged proteins specifically in intestinal epithelial cells. (A–A″) mCherry-RME-8 colocalizes with GFP-SNX-1. Arrowheads indicate endosomes labelled by both GFP-SNX-1 and mCherry-RME-8. (B–B″) mCherry-SNX-1 colocalizes with early endosome marker GFP-RAB-5. Arrowheads indicate endosomes labelled by both GFP-RAB-5 and mCherry-SNX-1. (C–C″) mCherry-SNX-1 does not colocalize well with Golgi marker MANS-GFP. Arrowheads and the inset indicate mCherry-SNX-1 positive endosomes juxtaposed to MANS-GFP-labelled Golgi ministacks. (D–D″) mCherry-RME-8 colocalizes with early endosome marker GFP-RAB-5. Arrowheads indicate endosomes labelled by both GFP-RAB-5 and mCherry-RME-8. (E–E″) mCherry-RME-8 does not colocalize well with Golgi marker MANS-GFP. Arrowheads and the inset indicate mCherry-RME-8 positive endosomes juxtaposed to MANS-GFP-labelled Golgi ministacks. Enlarged images (× 4) of boxed regions are shown in the insets. In each image, autofluorescent lysosome-like organelles can be seen in all three channels with the strongest signal in blue, whereas GFP appears only in the green channel and mCherry only in the red channel. Signals observed in the green or red channels that do not overlap with signals in the blue channel are considered bone fide GFP or mCherry signals, respectively. Scale bar represents 10 μm.

Clathrin accumulates on endosomes in animals lacking RME-8, SNX-1, or HSP-1/Hsc70. Panels show confocal micrographs of intestinally expressed GFP-tagged clathrin heavy chain (GFP-CHC-1) in intact living animals (A–D). Note the increased GFP-CHC-1 intensity in rme-8(b1023), snx-1(tm847), and hsp-1(RNAi) animals. (E) Bar graph indicates average GFP-CHC-1 fluorescence intensity in the indicated genetic backgrounds. Asterisks indicate a significant difference in the one-tailed Student's t-test (P<0.01). P-values for GFP-CHC-1 were 4.27 × 10^–17 for wild type (WT) versus rme-8(b1023), 5.92 × 10^–26 for WT versus snx-1(tm847) and 3.05 × 10^–19 for WT versus hsp-1(RNAi). For panels F–M″, images from deconvolved 3D image stacks are shown. All images were acquired in intact living animals expressing GFP and mCherry-tagged proteins specifically in intestinal epithelial cells. Autofluorescent lysosome-like organelles are shown in blue. GFP-CHC-1 could be visualized on WT and mutant mCherry-RAB-5-labelled endosomes in WT and mutants. Arrowheads indicated colocalization of the GFP-CHC-1 and mCherry-RAB-5 signals (F–I). GFP-CHC-1 could be visualized on WT and snx-1 mutant mCherry-RME-8-labelled endosomes. Arrowheads indicated colocalization of the GFP-CHC-1 and mCherry-RME-8 signals (J–K″). GFP-CHC-1 could be visualized on WT and rme-8 mutant mCherry-SNX-1-labelled endosomes. Arrowheads indicated colocalization of the GFP-CHC-1 and mCherry-SNX-1 signals (L–M″). Scale bars represent 10 μm.