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. 2017 Feb;18(2):134-144.
doi: 10.1111/tra.12462. Epub 2017 Jan 16.

Distinct complexes of yeast Snx4 family SNX-BARs mediate retrograde trafficking of Snc1 and Atg27

Mengxiao Ma  1 , Christopher G Burd  1 , Richard J Chi  2
Affiliations

Distinct complexes of yeast Snx4 family SNX-BARs mediate retrograde trafficking of Snc1 and Atg27

Mengxiao Ma et al. Traffic. 2017 Feb.

Abstract

The yeast SNX4 sub-family of sorting nexin containing a Bin-Amphiphysin-Rvs domain (SNX-BAR) proteins, Snx4/Atg24, Snx41 and Atg20/Snx42, are required for endocytic recycling and selective autophagy. Here, we show that Snx4 forms 2 functionally distinct heterodimers: Snx4-Atg20 and Snx4-Snx41. Each heterodimer coats an endosome-derived tubule that mediates retrograde sorting of distinct cargo; the v-SNARE, Snc1, is a cargo of the Snx4-Atg20 pathway, and Snx4-Snx41 mediates retrograde sorting of Atg27, an integral membrane protein implicated in selective autophagy. Live cell imaging of individual endosomes shows that Snx4 and the Vps5-Vps17 retromer SNX-BAR heterodimer operate concurrently on a maturing endosome. Consistent with this, the yeast dynamin family protein, Vps1, which was previously shown to promote fission of retromer-coated tubules, promotes fission of Snx4-Atg20 coated tubules. The results indicate that the yeast SNX-BAR proteins coat 3 distinct types of endosome-derived carriers that mediate endosome-to-Golgi retrograde trafficking.

Keywords: SNX-BAR; Atg27; Vps1; autophagy; endosome; retromer.

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Figures

Figure 1
Figure 1. Snx4 is required for organelle targeting of Snx41 and Atg20
(A) Micrographs showing cells expressing Snx4, Snx41 and Atg20 as C-terminal tandem GFP fusion proteins in wild-type and the indicated mutant cells are shown. (B) Micrographs showing Snx4-2xRFP colocalization with Snx41-2xGFP and Atg20-2xGFP, Pearson’s correlations are Rave=0.49 (n=31), Rave=0.52 (n=31), respectively. Maximum projections are shown. The scale bar indicates 5 μm.
Figure 2
Figure 2. Snx4 family proteins coat endosome-derived membrane tubules
(A) Micrographs of Snx4-2xGFP, Atg20-2xGFP and Snx41-2xGFP decorated endosomes captured by time-lapse fluorescence deconvolution microscopy. Galleries are representative of SNX-BAR coated endosomes with an associated tubular endosomal network. Arrowheads point to tubules of interest and open arrows (<) indicate apparent fission events. Images were captured at a single focal plane and acquired at 200 msec intervals. (B) Micrographs showing cells expressing Mup1-mTagBFP2, Snx4-2xGFP, Vps17-2xRFP in methionine depleted media. Within 15 minutes of the addition of 20 μg/ml methionine, a subset of Snx4 and Vps17 (a retromer subunit) endosomes accumulated Mup1. Pearson’s correlations of Snx4 and Vps17 is Rave=0.5 (n=20) (C) Micrographs showing distinct tubules emanating from Mup1-mTagBFP2, Snx4-2xGFP, Vps17-2xRFP endosomes. Endosomes were captured by three-color time-lapse fluorescence deconvolution microscopy. An example of a tubule decorated by only Snx4 is shown at 2.1 sec, a tubule decorated by only Vps17-2xRFP is shown at 6.3 secs. For visual contrast, individual colors are shown in gray scale. (D) Quantitation of the different types of SNX-BAR-coated tubules. Of 100 tubules analyzed, 40% were decorated with only Snx4, 57% were decorated with only Vps17, and 3% were decorated with both.
Figure 3
Figure 3. SNX-BARs and an ESCRT pathway component define distinct domains of the endosome membrane
(A) Micrographs showing Did2-mTagBFP2, an ESCRT pathway component, decorating a distinct domain of a common double labeled Vps17-mTagRuby2, Snx4-2xGFP endosome. The graph shows an example line scan of a typical endosome (inset). (B) Micrographs showing three-color time lapse fluorescence microscopy of an endosome. Did2 remains on the endosome while the levels of Snx4 and Vps17 gradually decrease; color-coded lines depict presence of indicated proteins in each frame. The scale bars in insets indicate 1 μm; the scale bar in whole cell micrographs indicates 5 μm.
Figure 4
Figure 4. Vps1 dynamin-related GTPase is required for fission of Snx4 coated endosomal transport carriers
(A) Micrographs of cells expressing Snx4-2xGFP and Vp17-2xRFP were visualized in wild-type and vps1Δ cells. Maximum projections of deconvolved Z stacks are shown. The scale bar indicates 5 μm. (B) A time lapse gallery of Snx4-2xGFP and Vps17-2xRFP coated endosomes in vps1Δ cells by time-lapse fluorescence deconvolution microscopy is shown. Arrows highlight a tubule of interest in each time-lapse series. In vps1Δ cells, Snx4 endosomal tubules are elongated and undergo fission less frequently. (C) Left Graph, Tubule lifetimes were calculated for Snx4-2xGFP or Vps17-2xRFP coated tubules. Snx4 (p<0.0001) and Vps17 (p=0.0005) tubule lifetimes were increased in vps1Δ cells; Right Graph, Endosome density (puncta/cell) was calculated by using optical Z-sections (0.30 um) for Snx4-2XGFP and Vps17-2xRFPs in wildtype and vps1Δ cells. Both Snx4 (p<0.0001) and Vps17 (p<0.0001) endosomes are increased in vps1Δ cells. Standard deviations are indicated and p values were calculated using paired Student’s t-test.
Figure 5
Figure 5. Snx4 and Atg20, but not Snx41, are required for plasma membrane recycling of Snc1
(A) Micrographs of ectopically expressed GFP-Snc1 in wild-type and mutant cells are shown. A single Z section from the approximate middle of each cell is shown for both fluorescent and DIC channels. (B) Cell lysates from indicated stains were probed with anti-GFP and the amount of free GFP was determined by quantitative western blot analysis. The positions of molecular mass (kDa) markers are indicated.
Figure 6
Figure 6. Snx4 and Snx41 are required to prevent accumulation of Atg27-ΔYSAV-2xGFP on the vacuole membrane
(A) Micrographs of cells expressing Atg27-ΔYSAV-2xGFP in wild-type, snx4Δ (B), snx41Δ (C), and atg20Δ (D) cells. Cells were grown to early log phase incubated with FM4-64 as described and imaged. Approximate middle slices of deconvolved Z stacks are shown. Vacuoles are identified with arrows. The scale bar indicates 5 μm. (E) Quantification of percent of cells with vacuolar membrane localization of Atg27-ΔYSAV-2xGFP as measured by continuous colocalization of Atg27-ΔYSAV-2xGFP with FM4-64. Error bars indicate SEM of three independent experiments.
Figure 7
Figure 7. Atg27 fails to mobilize from the vacuole membrane in response to rapamycin treatment in snx4Δ and snx41Δ cells
(A) Micrographs of cells expressing Atg27-link-2xGFP in wild-type, snx4Δ (B), and snx41Δ (C) cells. Cells were grown to early log phase and incubated with rapamycin (0.2μg/mL) for 4 hr to induce autophagy and labeled with FM4-64 dye as described. Approximate middle slices of deconvolved Z stacks are shown. Vacuoles are identified with arrows. The scale bar indicates 5 μm. (D) Quantification of percent of cells with vacuolar membrane localization of Atg27-link-2xGFP as measured by continuous colocalization of Atg27-link-2xGFP with FM4-64. Error bars indicate SEM of three independent experiments.
Figure 8
Figure 8. Summary of SNX-BAR sorting pathways in the yeast endosomal system
The endosomal maturation pathway is depicted. We propose that the Snx4 family proteins function concurrently with retromer SNX-BARs on an endosome to sort distinct cargo proteins. Snx4 dimerizes with Snx41 or Atg20, each of which confer distinct cargo specificities. Snx4-Atg20 functions on the Snc1 v-SNARE recycling pathway to transport Snc1 to the Golgi, and Snx4-Snx41 functions to transport Atg27 to the Golgi where it is then transported via the AP3 pathway to the vacuolar membrane. We speculate that Snx4-Atg20 and Snx4-Snx41 heterodimers coat distinct carriers.
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