Cargo induces retromer-mediated membrane remodeling on membranes.

Endosomes serve as a central sorting station of lipids and proteins that arrive via vesicular carrier from the plasma membrane and the Golgi complex. At the endosome, retromer complexes sort selected receptors and membrane proteins into tubules or vesicles that bud off the endosome. The mature endosome finally fuses with the lysosome. Retromer complexes consist of a cargo selection complex (CSC) and a membrane remodeling part (sorting nexin [SNX]-Bin/amphiphysin/Rvs [BAR], or Snx3 in yeast) and different assemblies of retromer mediate recycling of different cargoes. Due to this complexity, the exact order of events that results in carrier formation is not yet understood. Here, we reconstituted this process on giant unilamellar vesicles together with purified retromer complexes from yeast and selected cargoes. Our data reveal that the membrane remodeling activity of both Snx3 and the SNX-BAR complex is strongly reduced at low concentrations, which can be reactivated by CSC. At even lower concentrations, these complexes still associate with membranes, but only remodel membranes in the presence of their specific cargoes. Our data thus favor a simple model, where cargo functions as a specific trigger of retromer-mediated sorting on endosomes.

Lipid trafficking by yeast Snx4 family SNX-BAR proteins promotes autophagy and vacuole membrane fusion.

Cargo-selective and nonselective autophagy pathways employ a common core autophagy machinery that directs biogenesis of an autophagosome that eventually fuses with the lysosome to mediate turnover of macromolecules. In yeast ( Saccharomyces cerevisiae) cells, several selective autophagy pathways fail in cells lacking the dimeric Snx4/Atg24 and Atg20/Snx42 sorting nexins containing a BAR domain (SNX-BARs), which function as coat proteins of endosome-derived retrograde transport carriers. It is unclear whether endosomal sorting by Snx4 proteins contributes to autophagy. Cells lacking Snx4 display a deficiency in starvation induced, nonselective autophagy that is severely exacerbated by ablation of mitochondrial phosphatidylethanolamine synthesis. Under these conditions, phosphatidylserine accumulates in the membranes of the endosome and vacuole, autophagy intermediates accumulate within the cytoplasm, and homotypic vacuole fusion is impaired. The Snx4-Atg20 dimer displays preference for binding and remodeling of phosphatidylserine-containing membrane in vitro, suggesting that Snx4-Atg20-coated carriers export phosphatidylserine-rich membrane from the endosome. Autophagy and vacuole fusion are restored by increasing phosphatidylethanolamine biosynthesis via alternative pathways, indicating that retrograde sorting by the Snx4 family sorting nexins maintains glycerophospholipid homeostasis required for autophagy and fusion competence of the vacuole membrane.

Retromer-driven membrane tubulation separates endosomal recycling from Rab7/Ypt7-dependent fusion.

Endosomes are the major protein-sorting hubs of the endocytic pathway. They sort proteins destined for degradation into internal vesicles while in parallel recycling receptors via tubular carriers back to the Golgi. Tubule formation depends on the Rab7/Ypt7-interacting retromer complex, consisting of the sorting nexin dimer (SNX-BAR) and the trimeric cargo selection complex (CSC). Fusion of mature endosomes with the lysosome-like vacuole also requires Rab7/Ypt7. Here we solve a major problem in understanding this dual function of endosomal Rab7/Ypt7, using a fully reconstituted system, including purified, full-length yeast SNX-BAR and CSC, whose overall structure we present. We reveal that the membrane-active SNX-BAR complex displaces Ypt7 from cargo-bound CSC during formation of recycling tubules. This explains how a single Rab can coordinate recycling and fusion on endosomes.

STX13 regulates cargo delivery from recycling endosomes during melanosome biogenesis.

Melanosomes are a class of lysosome-related organelles produced by melanocytes. Biogenesis of melanosomes requires the transport of melanin-synthesizing enzymes from tubular recycling endosomes to maturing melanosomes. The SNARE proteins involved in these transport or fusion steps have been poorly studied. We found that depletion of syntaxin 13 (STX13, also known as STX12), a recycling endosomal Qa-SNARE, inhibits pigment granule maturation in melanocytes by rerouting the melanosomal proteins such as TYR and TYRP1 to lysosomes. Furthermore, live-cell imaging and electron microscopy studies showed that STX13 co-distributed with melanosomal cargo in the tubular-vesicular endosomes that are closely associated with the maturing melanosomes. STX family proteins contain an N-terminal regulatory domain, and deletion of this domain in STX13 increases both the SNARE activity in vivo and melanosome cargo transport and pigmentation, suggesting that STX13 acts as a fusion SNARE in melanosomal trafficking pathways. In addition, STX13-dependent cargo transport requires the melanosomal R-SNARE VAMP7, and its silencing blocks the melanosome maturation, reflecting a defect in endosome-melanosome fusion. Moreover, we show mutual dependency between STX13 and VAMP7 in regulating their localization for efficient cargo delivery to melanosomes.