A conformational switch driven by phosphorylation regulates the activity of the evolutionarily conserved SNARE Ykt6

Ykt6 is a soluble N-ethylmaleimide sensitive factor activating protein receptor (SNARE) critically involved in diverse vesicular fusion pathways. While most SNAREs rely on transmembrane domains for their activity, Ykt6 dynamically cycles between the cytosol and membrane-bound compartments where it is active. The mechanism that regulates these transitions and allows Ykt6 to achieve specificity toward vesicular pathways is unknown. Using a Parkinson’s disease (PD) model, we found that Ykt6 is phosphorylated at an evolutionarily conserved site which is regulated by Ca²⁺ signaling. Through a multidisciplinary approach, we show that phosphorylation triggers a conformational change that allows Ykt6 to switch from a closed cytosolic to an open membrane-bound form. In the phosphorylated open form, the spectrum of protein interactions changes, leading to defects in both the secretory and autophagy pathways, enhancing toxicity in PD models. Our studies reveal a mechanism by which Ykt6 conformation and activity are regulated with potential implications for PD.

Membrane fusion represents the final stage in vesicle trafficking and is largely driven by the soluble N-ethylmeleimide sensitive factor activating protein receptor (SNARE) proteins, which contribute to the specificity of the trafficking event (1, 2). Ykt6 is an essential R-SNARE and one of the most highly conserved SNAREs in all eukaryotes (3, 4). Ykt6 plays a key role in numerous vesicular transport pathways in yeast and in mammalian cells: 1) the secretory pathways, which are endoplasmic reticulum (ER) to the Golgi apparatus (Golgi), intra-Golgi, Golgi–ER retrieval pathways, and in the constitutive transport from the Golgi to the plasma membrane (3, 5, 6); 2) the endocytic pathways, which are between the Golgi and the vacuole/lysosome (7, 8) and endosomes to exosomes (9); and 3) the macroautophagy pathway (hereafter referred to as autophagy) (10–12). To date, the mechanism(s) underlying Ykt6 recruitment to distinct vesicular pathways remains unresolved.Unlike most SNAREs, Ykt6 contains a C-terminal lipid anchor motif which can be reversibly either palmitoylated (7, 13, 14) or geranylgeranylated (15) at cysteine 194 and permanently farnesylated at cysteine 195 (16). The reversible nature of palmitoylation and/or geranylgeranylation has been proposed as a mechanism to regulate Ykt6 membrane association, allowing it to cycle between the cytosol and membrane-bound compartments (14, 17). While lipid modifications are crucial to regulate Ykt6 membrane association, NMR and crystallography studies demonstrated that activation of Ykt6 is far more complex (18). When in the cytosol, Ykt6 forms a closed conformation whereby the N-terminal regulatory longin domain folds back onto the SNARE domain stabilized by hydrophobic interactions (18–20). Deletion of the longin domain, or substitution of a hydrophobic residue to a negatively charged one within the Ykt6 longin domain (F42E), causes the longin domain to dissociate from the SNARE domain and relocalize Ykt6 to both the plasma membrane and Golgi (14, 18). These data have led to a model whereby, for Ykt6 to be active and membrane associated, the longin and SNARE domains must separate to facilitate the open conformation. Once in the open conformation, Ykt6 can be palmitoylated, providing its membrane stability and interactions with its SNARE partners (14, 19). Whether this is the molecular mechanism that activates Ykt6, as well as the in vivo physiological triggers, remains to be shown.Misfolding of α-synuclein (α-syn) is the pathological hallmark of both familial and sporadic Parkinson’s disease (PD) (21–24). α-Syn triggers high levels of cytosolic Ca²⁺ and the activation of calcineurin (CaN), a Ca²⁺-dependent phosphatase (25, 26). Using an unbiased phosphoproteomic approach in a yeast model for α-syn toxicity, we previously found endogenous Ykt6 phosphorylated and regulated by CaN (27) (Fig. 1A). Ykt6 itself has been implicated in α-syn pathobiology as Ykt6 overexpression overcomes the secretory trafficking deficits caused by α-syn and protects against cell death (28, 29). Whether Ykt6 regulation by phosphorylation contributes to Ykt6 deficits under α-syn toxicity remains to be determined.Open in a separate windowFig. 1.Evolutionarily conserved phosphorylation site within human Ykt6 SNARE domain (S174) is sensitive to calcineurin, evolutionarily conserved in yeast and in humans, and is a critical determinant for its intracellular localization. (A) Fold phosphorylation of the indicated peptide from endogenous yeast Ykt6 detected by shotgun phosphoproteomics after correction for protein abundance from control yeast cells and yeast cells with high levels of Ca²⁺ (driven by overexpression of α-syn) with either WT or knockout for calcineurin (∆CaN) and knockout for the modulator of calcineurin (∆FKBP12). The identified phosphorylation sites are highlighted in red. Data from triplicate samples were pulled together for illustrated analysis (28). Data from ref. 27. (B) Alignment of Ykt6 sequences across species. Highlighted in pink is the identified phosphorylation site conserved across evolution. Arrows depicted represent Ykt6 zero-layer arginine (white) and additional CaN-sensitive sites identified in phosphoproteomic screen (red is human, black is yeast). (C) Fold phosphorylation of the indicated human Ykt6 peptide from HEK293T cells as detected by iTRAQ MS. Prior to GFP-Ykt6 immunoprecipitation, cells were treated for 30 min with the Ca²⁺ ionophore ionomycin (1 μM) and cotreated with calcineurin-specific inhibitor tacrolimus (1 μM). (D) Representative IF images of transiently transfected HeLa cells with GFP-tagged wild-type (WT) or phosphomimetic mutant of Ykt6 (S174D). Cells were treated with ionomycin (1 μM) and/or tacrolimus (1 μM) for 30 min. Nuclei (blue) are stained with DAPI. (Scale bar, 10 μm.) (E) Quantification of cells with GFP plasma membrane localization as shown in A. n = 3; *P < 0.05, ***P < 0.001, ****P < 0.0001. One-way ANOVA, uncorrected Fisher’s least significance difference (LSD) test. (F) Quantification of Ykt6 on membrane fractions (Na⁺K+)/Ykt6 on cytosol fractions (tubulin) from Western blots after cell fractionation (see Dataset S1); n = 3. (G, H) Quantification of GFP-WT Ykt6 (G) or GFP-S174D Ykt6 (H) on PM fraction (see Materials and Methods for details) of HEK293T cotransfected with the indicated Flag-tagged kinases. (I) Quantification of endogenous Ykt6 membrane (Na⁺K+)/Ykt6 cytosol (tubulin) fraction from Western blots after cell fractionation of HEK293T cells transfected with the indicated Flag-tagged kinases; n = 3 (see SI Appendix, Fig. S5D). Stats for (F–I) **P < 0.05, ***P < 0.001, ****P < 0.0001. One-way ANOVA, uncorrected Fisher’s LSD test. All comparisons to dimethyl sulfoxide (dmso) control of Flag-transfected dmso. (J) 40× confocal images after IF for YKT6 (green) and FLAG-tagged candidate kinases (red) of HEK293T cells cotransfected with GFP-WT-Ykt6 and the indicated Flag-tagged kinase. Single Z-frame images were channel merged and background subtracted before processed for quantitative analysis. Individual representative cells were chosen from each condition to better present changes in intracellular localization of YKT6. (Scale bar, 10 μm.) n = 3; ∼600 cells total.Here, we report that phosphorylation at the evolutionarily conserved site S174 within the Ykt6 SNARE domain drives an intramolecular rearrangement mediating the conversion from a closed cytosolic to an open membrane-bound state. Using an unbiased high-content kinase screening assay, we found that protein kinase C iota type (PRKCi) regulates Ykt6 phosphorylation and membrane association. Furthermore, we demonstrate that phosphorylation is sensitive to CaN and a key determinant of the binding affinity and specificity for various protein interactions. Phospho-dependent interactions of Ykt6 have functional consequences in two cellular activities in which Ykt6 has been shown to play a role: the secretory and autophagy pathways. Moreover, we show the biological importance of the evolutionarily conserved phosphosite under physiological and α-syn toxic conditions in yeast and Caenorhabditis elegans models of PD. Taken together, our results provide a mechanistic insight into the regulation of Ykt6 and its cellular activities with implications for PD, wherein malfunctions in Ykt6, autophagy, and the secretory pathway have been attributed (28–30).     

A study on the correlation of blood and vitreous humour alcohol levels in the late absorption and elimination phases

By using the urine:blood alcohol level ratio as the indicator, the correlation of blood alcohol level (B) and vitreous humour alcohol level (V) in the late absorption and elimination phases was studied. It was found to be good (r = 0.98) and B = 0.76V + 4.7. It is suggested that this equation can safely be used to estimate the minimum blood alcohol level where cadaveric blood is unsuitable or unavailable for analysis and that the B/V ratio can be used to infer the phase in which death occurred where urine is not available.     

The initial and subsequent inflammatory events during calcium oxalate lithiasis

BackgroundCrystallization is believed to be the initiation step of urolithiasis, even though it is unknown where inside the nephron the first crystal nucleation occurs.MethodsDirect nucleation of calcium oxalate and subsequent events including crystal retention, cellular damage, endocytosis, and hyaluronan (HA) expression, were tested in a two-compartment culture system with intact human proximal tubular HK-2 cell monolayer.ResultsCalcium oxalate dihydrate (COD) was nucleated and bound onto the apical surface of the HK-2 cells under hypercalciuric and hyperoxaluric conditions. These cells displayed mild cellular damage and internalized some of the adhered crystals within 18 h post-COD-exposure, as revealed by electron microscopy. Prolonged incubation in complete medium caused significant damage to disrupt the monolayer integrity. Furthermore, hyaluronan disaccharides were detected in the harvested media, and were associated with HAS-3 mRNA expression.ConclusionHuman proximal cells were able to internalize COD crystals which nucleated directly onto the apical surface, subsequently triggering cellular damage and HAS-3 specific hyaluronan synthesis as an inflammatory response. The proximal tubule cells here demonstrate that it plays an important role in facilitating urolithiasis via endocytosis and creating an inflammatory environment whereby free hyaluronan in tubular fluid can act as crystal-binding molecule at the later segments of distal and collecting tubules.     

Development of biobased emulsions for postharvest citrus fruit preservation

Penicillium digitatum is the causal agent for citrus green mold which generates 90% of post-harvest losses in Mediterranean countries. The use of a green seaweed extract to fight this fungal pathogen could represent an ecofriendly alternative to chemical fungicides. We propose to formulate an aqueous extract from Ulva lactuca (8.3%) in an emulsion for the preventive treatment of oranges. New biobased emulsifiers, alkyl aconitates, were synthesized according to a green process and were characterized by their hydrophilic and lipophilic balance (HLB) and their critical micelle concentration (CMC). These mono-, di and tri-dodecyl aconitates (8%) were effective in stabilizing an oil-in-water emulsion including the natural U. lactuca extract. The viscosities of resulting emulsions helped to form a film on the orange surface. Several in vitro microbiological tests towards Penicillium digitatum showed interesting anti-fungal activities for the new ingredients, as well as for the resulting emulsions. In-vivo tests also confirmed that mono-, di- and tri-docedyl aconitates could contribute to limit the development of P. digitatum on peel surface. Thus, the compositions of two ecofriendly emulsions combining a natural extract and a biobased emulsifier seem adapted to improve oranges preservation.