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. 2007 Nov 20;17(22):1913-24.
doi: 10.1016/j.cub.2007.10.045. Epub 2007 Nov 8.

A novel requirement for C. elegans Alix/ALX-1 in RME-1-mediated membrane transport

Anbing Shi  1 , Saumya Pant, Zita Balklava, Carlos Chih-Hsiung Chen, Vanesa Figueroa, Barth D Grant
Affiliations

A novel requirement for C. elegans Alix/ALX-1 in RME-1-mediated membrane transport

Anbing Shi et al. Curr Biol. .

Abstract

Background: Alix/Bro1p family proteins have recently been identified as important components of multivesicular endosomes (MVEs) and are involved in the sorting of endocytosed integral membrane proteins, interacting with components of the ESCRT complex, the unconventional phospholipid LBPA, and other known endocytosis regulators. During infection, Alix can be co-opted by enveloped retroviruses, including HIV, providing an important function during virus budding from the plasma membrane. In addition, Alix is associated with the actin cytoskeleton and might regulate cytoskeletal dynamics.

Results: Here we demonstrate a novel physical interaction between the only apparent Alix/Bro1p family protein in C. elegans, ALX-1, and a key regulator of receptor recycling from endosomes to the plasma membrane, called RME-1. The analysis of alx-1 mutants indicates that ALX-1 is required for the endocytic recycling of specific basolateral cargo in the C. elegans intestine, a pathway previously defined by the analysis of rme-1 mutants. The expression of truncated human Alix in HeLa cells disrupts the recycling of major histocompatibility complex class I, a known Ehd1/RME-1-dependent transport step, suggesting the phylogenetic conservation of this function. We show that the interaction of ALX-1 with RME-1 in C. elegans, mediated by RME-1/YPSL and ALX-1/NPF motifs, is required for this recycling process. In the C. elegans intestine, ALX-1 localizes to both recycling endosomes and MVEs, but the ALX-1/RME-1 interaction appears to be dispensable for ALX-1 function in MVEs and/or late endosomes.

Conclusions: This work provides the first demonstration of a requirement for an Alix/Bro1p family member in the endocytic recycling pathway in association with the recycling regulator RME-1.

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Figures

Figure 1
Figure 1. RME-1 Interacts with ALX-1
Quantitative yeast two-hybrid beta-galactosidase assays show that the ALX-1 C-terminal NPF motif contributes to the interaction with RME-1, while the central domain (aa 365-752) of ALX-1 contributes the main binding surface, interacting with RME-1 through its C-terminal YPSL sequence. Y-axis is labeled in Miller Units.
Figure 2
Figure 2. ALX-1 is Broadly Expressed in C.elegans
Expression of a GFP-ALX-1 transgene driven by the alx-1 promoter is indicated in (A-F). (A) Intestine, arrowheads indicate cytoplasmic puncta. (B) Pharynx is indicated by arrows and nerve ring indicated by arrowheads. (C) Arrow indicates spermatheca and arrowhead indicates cytoplasmic puncta. (D) Coelomocyte, cytoplasmic puncta (arrowheads). (E) Nerve cord (arrowheads) and body-wall muscle (arrow). (F) Hypodermis, cytoplasmic puncta (arrowheads). Scale bars represent 10 µm.
Figure 3
Figure 3. ALX-1 Associates with Two Types of Endosomes in the Intestine
(A-C) mCherry-ALX-1 colocalizes with GFP-RME-1 on basolateral recycling endosomes. Arrowheads indicate endosomes labeled by both GFP-RME-1 and mCherry-ALX-1. (D-F) Some mCherry-ALX-1 also colocalizes with MVE marker GFP-HGRS-1, mostly in the medial and apical cytoplasm. Arrowheads indicate puncta labeled by both mCherry-ALX-1 and GFP-HGRS-1. (G-I) mCherry-RME-1 and GFP-HGRS-1 label different endosome types. Virtually no overlap was observed between mCherry-RME-1 and GFP-HGRS-1. In each image autofluorescent lysosomes 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 5 µm.
Figure 4
Figure 4. rme-1 Mutants Accumulate Abnormally Numerous GFP-ALX-1 Labeled Endosomes
Confocal images of wild-type animals (A) and rme-1(b1045) mutant animals (B) showing GFP-ALX-1 labeled endosomes in the intestine. Arrowheads indicate GFP-ALX-1 puncta. (C) Quantification of endosome number in wild type animals and mutants as visualized by GFP-ALX-1. Error bars represent standard deviations from the mean (n=18 each, 6 animals of each genotype sampled in three different regions of each intestine). Asterisks indicate a significant difference in the one-tailed Student's t-test (p<0.01). Scale bar represents 10 µm.
Figure 5
Figure 5. Abnormal Trafficking of Recycling Cargo in alx-1 Mutant C. elegans and in HeLa Cells Expressing Truncated Alix
(A-B) Localization and morphology of hTfR-GFP, a clathrin-dependent cargo protein, appears unchanged in alx-1(gk275) mutants compared to wild-type animals. (C-D) hTAC-GFP, a cargo protein internalized independently of clathrin, becomes trapped in endosomal structures of alx-1 mutants. Arrowheads indicate punctate and tubular endosomes labeled by hTAC-GFP in the intestine. (E) Quantification of cargo-labeled puncta number. Error bars represent standard deviations from the mean (n=18 each, 6 animals of each genotype sampled in three different regions of each intestine). (F-I) Representative images showing anti-MHCI labeling in control HeLa cells transfected with RFP expression plasmid only (F, H) or for cells co-transfected with RFP and truncated Alix expression plasmids (G, I). The first pair of images (F, G) show anti-MHCI uptake after 30 min incubation. The second pair of images (H, I) show surface MHCI after 30 minutes of recycling (H, I). Cells that were negative for the RFP signal are marked with asterisks. In co-transfection experiments these RFP-negative cells may or may not express Alix(467-869) (see Methods) and were therefore not included in the quantification. Conversely nearly all RFP expressing cells were found to also express FLAG-Alix(467-869) in control experiments. Therefore only RFP expressing cells were quantified for anti-MHCI uptake or recycling. (J-K) The amount of MHCI antibody internalized after a 30 min pulse, or recycled after a 30 min chase, is shown as a ratio relative to control cells (see Methods). MHCI antibody recycling (K), but not MHCI antibody uptake (J), was impaired by expression of truncated FLAG-Alix(467-869). The asterisk in panel K indicates a significant difference in the one-tailed Student's t-test (p=0.004). Scale bars represent 10 µm in (A-D) and 20 µm in (F-I).
Figure 6
Figure 6. Altered Endosome Populations in alx-1 Mutants
(A-B) GFP-RME-1 labeled recycling endosomes increase in number in alx-1 mutant intestinal cells. Arrowheads indicate punctate and tubular endosomes labeled by GFP-RME-1. (C-D) GFP-HGRS-1 positive endosomes increase in number in alx-1 mutant intestinal cells. Arrowheads indicate punctate endosomes labeled by GFP-HGRS-1. (E-H) GFP-RAB-7 and LMP-1-GFP labeled late endosomes appear enlarged and aggregated in alx-1(gk275) mutant intestinal cells. (J-K) Quantification of GFP-labeled puncta number and puncta size. Error bars represent standard deviations from the mean (n=18 each, 6 animals of each genotype sampled in three different regions of each intestine). The asterisks indicate a significant difference in the one-tailed Student's t-test (p<0.01). Scale bars represent 10 µm.
Figure 7
Figure 7. (A-D) Syndapin/SDPN-1-GFP Localization is Abnormal in alx-1 Mutant and rme-1 Mutant Intestinal Cells
(A) Confocal images of wild-type animals show abundant SDPN-1-GFP labeled basolateral punctate and tubular endosomes. (B) In alx-1 mutant animals most basolateral SDPN-1-GFP positive structures are missing, and most of the remaining SDPN-1-GFP signal appears diffuse. (C) In rme-1 mutant animals basolateral SDPN-1-GFP labeled structures are fewer, and many of those that remain are enlarged. (D) alx-1(gk275); rme-1(RNAi) animals lack most basolateral SDPN-1-GFP labeled structures like alx-1 mutant animals, but the remaining structures appear enlarged. Arrowheads indicate endosomes labeled by SDPN-1-GFP. Scale bar represents 10 μm. (E-H) Rescue Experiments Genetically Separate ALX-1 Functions and Indicate a Requirement for the ALX-1/RME-1 Interaction in vivo. Interaction defective mutant forms of RME-1 (ΔYPSL) or ALX-1 (ΔNPF), expressed as mCherry (MC) fusions, were compared to wild-type forms in their ability to rescue specific rme-1(b1045) or alx-1(gk275) mutant phenotypes in the intestine. One set of phenotypes that were assayed focused on recycling endosomes: abnormal accumulation of basolaterally recycling pseudocoelomic fluid in enlarged vacuoles/REs, intracellular recycling cargo hTAC-GFP accumulation, and basolateral SDPN-1-GFP recruitment/morphology. In addition, ALX-1(ΔNPF) was compared to intact ALX-1 in its ability to rescue alx-1 mutant associated defects in the degradative pathway: multi-vesicular endosome number, as assayed by GFP-HGRS-1, and late endosome size, as assayed by LMP-1-GFP. (E) Expression of either MC-tagged RME-1 or MC-tagged RME-1(ΔYPSL) rescues basolateral fluid accumulation in rme-1(b1045) mutants. (F) The last two bars in each group show the relative degree of rescue of rme-1(b1045) mutant defects achieved by expression of MC-tagged RME-1, or MC-tagged RME-1(ΔYPSL). Note that wild-type MC-RME-1 rescues both defects, while MC-RME-1(ΔYPSL) cannot rescue the rme-1(b1045) associated defects. (G-H) The last two bars in each graph show the relative degree of rescue of alx-1(gk275) mutant defects achieved by expression of MC-tagged ALX-1, or MC-tagged ALX-1(ΔNPF). Note that wild-type MC-ALX-1 rescues all defects, while MC-ALX-1(ΔNPF) can only rescue alx-1 associated phenotypes of the degradative pathway, LMP-1-GFP endosome size (G) and GFP-HGRS-1 puncta number (H), but cannot rescue phenotypes associated with the recycling pathway, hTAC-GFP and SDPN-1-GFP puncta number (H). Asterisks indicate a significant difference in the one-tailed Student's t-test (p<0.01).
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