The VINE complex is an endosomal VPS9-domain GEF and SNX-BAR coat
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Evaluation Summary:
The SNX-BAR family of sorting nexins are a diverse group of dimeric proteins that form tubules from endosomal membranes where they also select protein cargoes for incorporation into these transport carriers. The authors of this manuscript describe a new SNX-BAR complex in budding yeast, named the VINE complex, that uniquely harbors a guanine nucleotide exchange factor (GEF) domain for Rab5-related small GTP-binding proteins. The author's data implicate VINE in endosomal membrane remodeling events and the sorting of a vacuolar hydrolase receptor. The paper would be strengthened by some additional quantifications and by taking advantage of new modeling opportunities.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their name with the authors.)
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Abstract
Membrane trafficking pathways perform important roles in establishing and maintaining the endosomal network. Retrograde protein sorting from the endosome is promoted by conserved SNX-BAR-containing coat complexes including retromer which enrich cargo at tubular microdomains and generate transport carriers. In metazoans, retromer cooperates with VARP, a conserved VPS9-domain GEF, to direct an endosomal recycling pathway. The function of the yeast VARP homolog Vrl1 has been overlooked due to an inactivating mutation found in commonly studied strains. Here, we demonstrate that Vrl1 has features of a SNX-BAR coat protein and forms an obligate complex with Vin1, the paralog of the retromer SNX-BAR protein Vps5. Unique features in the Vin1 N-terminus allow Vrl1 to distinguish it from Vps5, thereby forming a complex that we have named VINE. The VINE complex occupies endosomal tubules and redistributes a conserved mannose 6-phosphate receptor-like protein from endosomes. We also find that membrane recruitment by Vin1 is essential for Vrl1 GEF activity, suggesting that VINE is a multifunctional coat complex that regulates trafficking and signaling events at the endosome.
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Evaluation Summary:
The SNX-BAR family of sorting nexins are a diverse group of dimeric proteins that form tubules from endosomal membranes where they also select protein cargoes for incorporation into these transport carriers. The authors of this manuscript describe a new SNX-BAR complex in budding yeast, named the VINE complex, that uniquely harbors a guanine nucleotide exchange factor (GEF) domain for Rab5-related small GTP-binding proteins. The author's data implicate VINE in endosomal membrane remodeling events and the sorting of a vacuolar hydrolase receptor. The paper would be strengthened by some additional quantifications and by taking advantage of new modeling opportunities.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested …
Evaluation Summary:
The SNX-BAR family of sorting nexins are a diverse group of dimeric proteins that form tubules from endosomal membranes where they also select protein cargoes for incorporation into these transport carriers. The authors of this manuscript describe a new SNX-BAR complex in budding yeast, named the VINE complex, that uniquely harbors a guanine nucleotide exchange factor (GEF) domain for Rab5-related small GTP-binding proteins. The author's data implicate VINE in endosomal membrane remodeling events and the sorting of a vacuolar hydrolase receptor. The paper would be strengthened by some additional quantifications and by taking advantage of new modeling opportunities.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
The manuscript by Shortill and colleagues describes a new sorting nexin heterodimer in budding yeast comprised of a Vps9 (Rab5-GEF) domain-containing, VARP-like protein (Vrl1) and a previously uncharacterized SNX-BAR protein the authors have named Vin1. The authors provide strong evidence that Vin1 and Vrl1 form a heterodimer, driven primarily by the ankyrin-repeat domain in Vrl1 binding to the N-terminal region in Vin1. This is a surprising result because both proteins have BAR domains and one would have assumed that these domains mediated heterodimerization as shown for other PX-BAR proteins. The protein-protein interaction studies were thorough and yielded novel insights into VINE structure and function.
The authors also find evidence that Vin1 interaction with phosphatidylinositol 3-phosphate …
Reviewer #1 (Public Review):
The manuscript by Shortill and colleagues describes a new sorting nexin heterodimer in budding yeast comprised of a Vps9 (Rab5-GEF) domain-containing, VARP-like protein (Vrl1) and a previously uncharacterized SNX-BAR protein the authors have named Vin1. The authors provide strong evidence that Vin1 and Vrl1 form a heterodimer, driven primarily by the ankyrin-repeat domain in Vrl1 binding to the N-terminal region in Vin1. This is a surprising result because both proteins have BAR domains and one would have assumed that these domains mediated heterodimerization as shown for other PX-BAR proteins. The protein-protein interaction studies were thorough and yielded novel insights into VINE structure and function.
The authors also find evidence that Vin1 interaction with phosphatidylinositol 3-phosphate (PI3P)-containing membranes facilitates the Vrl1/RabGEF-dependent activation of PI 3-kinase, suggesting a potential mechanism for amplifying PI3P production on endosomes or vacuoles. These conclusions are based on indirect readouts (such as GFP-FYVE or Vps26 endosomal localization are used as a proxy of PI3P levels) and a few assumptions (there is good genetic support for the Vrl1 Rab-GEF activity but no direct biochemical evidence), but the approaches are standard for the field and support the conclusions well.
Finally, the authors also present evidence that the Vrl1-Vin1 complex sorts a mannose-6-phosphate receptor-like (Mrl1) protein in the endosomal system although the data supporting this conclusion is not as strong. The differences in Mrl1 localization between cells with or without Vrl1 are subtle and not quantified in a manner that would clearly indicate a mislocalization phenotype. The title, abstract and final model of the manuscript emphasize this role of the Vrl1-Vin1 complex in protein sorting. Therefore, additional supportive data on this point would significantly strengthen the manuscript.
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Reviewer #2 (Public Review):
This is an interesting study by Shortill and colleagues exploring the function of the yeast Vps9-domain family member Vrl1, building on previous work from the same lab identifying Vrl1 as a distant homologue of the mammalian retromer-interacting Rab GEF VARP. It presents an interesting combination of yeast genetics, biochemistry and imaging with molecular modelling using the AlphaFold2 machine learning algorithm to predict protein domain structures and protein-protein interactions and validation of these prediction in cells. Major findings include that (i) Vrl1 contains not only RabGEF and ankyrin-repeat domains but also PX and BAR domains with some homology to the retromer-interacting SNX-BAR proteins Vps5/Vps17, (ii) Vrl1 forms an obligate complex with another poorly studied SNX-BAR protein Vin1/Ykr078w …
Reviewer #2 (Public Review):
This is an interesting study by Shortill and colleagues exploring the function of the yeast Vps9-domain family member Vrl1, building on previous work from the same lab identifying Vrl1 as a distant homologue of the mammalian retromer-interacting Rab GEF VARP. It presents an interesting combination of yeast genetics, biochemistry and imaging with molecular modelling using the AlphaFold2 machine learning algorithm to predict protein domain structures and protein-protein interactions and validation of these prediction in cells. Major findings include that (i) Vrl1 contains not only RabGEF and ankyrin-repeat domains but also PX and BAR domains with some homology to the retromer-interacting SNX-BAR proteins Vps5/Vps17, (ii) Vrl1 forms an obligate complex with another poorly studied SNX-BAR protein Vin1/Ykr078w required for recruitment and activity at yeast endosomes, (iii) surprisingly, the interactions between Vrl1 and Vin1 are primarily mediated through Vin1 N-terminal sequences interacting with the AnkRD domain of Vrl1, and (iv) this complex is important for mediating tubulovesicular transport of the transmembrane cargo Mrl1. The authors conclude these proteins cooperate to regulate Rab activation and cargo transport at endosomes, and propose to name this the "VPS9 GEF-Interacting Sorting Nexin" or VINE complex. For context I have expertise in the structural biology and biochemistry of endosomal trafficking proteins and have some experience using AlphaFold2, but I do not have specific technical expertise in the yeast assays reported here.
The paper is well written, and I found the data to be convincing and supportive of the final conclusions. I did not find any major weaknesses, and I think with minor changes this will be of great interest to those in the field of membrane trafficking and the role of sorting nexin proteins in endosomal sorting. The use of AlphaFold2 also provides an interesting example of how this novel tool can be used to guide molecular and cellular studies of protein function.
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