Expression, Purification, and Liposome Binding of Budding Yeast SNX-BAR Heterodimers

The sorting nexins (SNXs), constitute a diverse family of molecules that play varied roles in membrane trafficking, cell signaling, membrane remodeling, organelle motility and autophagy. In particular, the SNX-Bin-Amphiphysin-Rvs (BAR) proteins, a SNX subfamily characterized by a C-terminal dimeric BAR lipid curvature domain and a conserved Phox-homology domain, are of great interest. In budding yeast, many SNX-BAR proteins have well-characterized endo-vacuolar trafficking roles. Phylogenetic analyses allowed us to identify an additional SNX-BAR protein, Vps501, with a novel endovacuolar role. We report that Vps501 uniquely localizes to the vacuolar membrane and has physical and genetic interactions with the SEA complex to regulate TORC1 inactivation. We found cells displayed a severe deficiency in starvation-induced/nonselective autophagy only when SEA complex subunits are ablated in combination with Vps501, indicating a cooperative role with the SEA complex during TORC1 signaling during autophagy induction. Additionally, we found the SEACIT complex becomes destabilized in vps501Δsea1Δ cells, which resulted in aberrant endosomal TORC1 activity and subsequent Atg13 hyperphosphorylation. We have also discovered that the vacuolar localization of Vps501 is dependent upon a direct interaction with Sea1 and a unique lipid binding specificity that is also required for its function.

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“…Snx4‐Atg20 were purified as previously described. 70 All purified protein concentrations were quantified by Brafford assay (Pierce).…”

Section: Methodsmentioning

confidence: 99%

Vps501, a novel vacuolar SNX‐BAR protein cooperates with the SEA complex to regulate TORC1 signaling

Goyal

Segarra

Nitika

et al. 2022

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Structural determinants specific for retromer protein sorting nexin 5 in regulating subcellular retrograde membrane trafficking

Chen¹,

Sun²,

Han³

et al. 2023

J Biomed Res

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The endosomal trafficking of signaling membrane proteins, such as receptors, transporters and channels, is mediated by the retromer-mediated sorting machinery, composed of a cargo-selective vacuolar protein sorting trimer and a membrane-deforming subunit of sorting nexin proteins. Recent studies have shown that the isoforms, sorting nexin 5 (SNX5) and SNX6, have played distinctive regulatory roles in retrograde membrane trafficking. However, the molecular insight determined functional differences within the proteins remains unclear. We reported that SNX5 and SNX6 had distinct binding affinity to the cargo protein vesicular monoamine transporter 2 (VMAT2). SNX5, but not SNX6, specifically interacted with VMAT2 through the Phox domain, which contains an alpha-helix binding motif. Using chimeric mutagenesis, we identified that several key residues within this domain were unique in SNX5, but not SNX6, and played an auxiliary role in its binding to VMAT2. Importantly, we generated a set of mutant SNX6, in which the corresponding key residues were mutated to those in SNX5. In addition to the gain in binding affinity to VMAT2, their overexpression functionally rescued the altered retrograde trafficking of VMAT2 induced by siRNA-mediated depletion of SNX5 . These data strongly suggest that SNX5 and SNX6 have different functions in retrograde membrane trafficking, which is determined by the different structural elements within the Phox domain of two proteins. Our work provides a new information on the role of SNX5 and SNX6 in the molecular regulation of retrograde membrane trafficking and vesicular membrane targeting in monoamine neurotransmission and neurological diseases.

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Expression, Purification, and Liposome Binding of Budding Yeast SNX-BAR Heterodimers

Cited by 2 publications

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Vps501, a novel vacuolar SNX‐BAR protein cooperates with the SEA complex to regulate TORC1 signaling

Vps501, a novel vacuolar SNX‐BAR protein cooperates with the SEA complex to regulate TORC1 signaling

Structural determinants specific for retromer protein sorting nexin 5 in regulating subcellular retrograde membrane trafficking

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