Spatial control of avidity regulates initiation and progression of selective autophagy

Autophagosomes form at the endoplasmic reticulum in mammals, and between the vacuole and the endoplasmic reticulum in yeast. However, the roles of these sites and the mechanisms regulating autophagosome formation are incompletely understood. Vac8 is required for autophagy and recruits the Atg1 kinase complex to the vacuole. Here we show that Vac8 acts as a central hub to nucleate the phagophore assembly site at the vacuolar membrane during selective autophagy. Vac8 directly recruits the cargo complex via the Atg11 scaffold. In addition, Vac8 recruits the phosphatidylinositol 3-kinase complex independently of autophagy. Cargo-dependent clustering and Vac8-dependent sequestering of these early autophagy factors, along with local Atg1 activation, promote phagophore assembly site assembly at the vacuole. Importantly, ectopic Vac8 redirects autophagosome formation to the nuclear membrane, indicating that the vacuolar membrane is not specifically required. We propose that multiple avidity-driven interactions drive the initiation and progression of selective autophagy.

The VINE complex is a VPS9–domain GEF–containing SNX–BAR coat involved in endosomal sorting

Membrane trafficking pathways perform important roles in establishing and maintaining the endolysosomal 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 an inactivating mutation 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 what we have named the VINE complex. VINE occupies endosomal tubules and promotes the delivery of a conserved mannose 6–phosphate receptor–like protein to the vacuolar membrane. In addition to sorting functions, 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.

Novel Nile Blue Analogue Stains Yeast Vacuolar Membrane, Endoplasmic Reticulum and Lipid Droplets, Inducing Cell Death through Vacuole Membrane Permeabilization

Phenoxazine derivatives such as Nile Blue analogues are assumed to be increasingly relevant in cell biology due to their fluorescence staining capabilities and antifungal and anticancer activities. However, the mechanisms underlying their effects remain poorly elucidated. Using S. cerevisiae as a eukaryotic model, we found that BaP1, a novel 5- and 9-N-substituted benzo[a]phenoxazine synthesized in our laboratory, when used in low concentrations, accumulates and stains the vacuolar membrane and the endoplasmic reticulum. In contrast, at higher concentrations, BaP1 stains lipid droplets and induces a regulated cell death process mediated by vacuolar membrane permeabilization. BaP1 also induced mitochondrial fragmentation and depolarization but did not lead to ROS accumulation, changes in intracellular Ca2+, or loss of plasma membrane integrity. Additionally, our results show that the cell death process is dependent on the vacuolar protease Pep4p and that the vacuole permeabilization results in its translocation from the vacuole to the cytosol. In addition, although nucleic acids are commonly described as targets of benzo[a]phenoxazines, we did not find any alterations at the DNA level. Our observations highlight BaP1 as a promising molecule for pharmacological application, using vacuole membrane permeabilization as a targeted approach.

MATE-Type Proteins Are Responsible for Isoflavone Transportation and Accumulation in Soybean Seeds

Soybeans are nutritionally important as human food and animal feed. Apart from the macronutrients such as proteins and oils, soybeans are also high in health-beneficial secondary metabolites and are uniquely enriched in isoflavones among food crops. Isoflavone biosynthesis has been relatively well characterized, but the mechanism of their transportation in soybean cells is largely unknown. Using the yeast model, we showed that GmMATE1 and GmMATE2 promoted the accumulation of isoflavones, mainly in the aglycone forms. Using the tobacco BrightYellow-2 (BY-2) cell model, GmMATE1 and GmMATE2 were found to be localized in the vacuolar membrane. Such subcellular localization supports the notion that GmMATE1 and GmMATE2 function by compartmentalizing isoflavones in the vacuole. Expression analyses showed that GmMATE1 was mainly expressed in the developing soybean pod. Soybean mutants defective in GmMATE1 had significantly reduced total seed isoflavone contents, whereas the overexpression of GmMATE1 in transgenic soybean promoted the accumulation of seed isoflavones. Our results showed that GmMATE1, and possibly also GmMATE2, are bona fide isoflavone transporters that promote the accumulation of isoflavones in soybean seeds.

Fusion of tethered membranes can be driven by Sec18/NSF and Sec17/αSNAP without HOPS

Yeast vacuolar membrane fusion has been reconstituted with R, Qa, Qb, and Qc-family SNAREs, Sec17/αSNAP, Sec18/NSF, and the hexameric HOPS complex. HOPS tethers membranes and catalyzes SNARE assembly into RQaQbQc trans-complexes which zipper through their SNARE domains to promote fusion. Previously, we demonstrated that Sec17 and Sec18 can bypass the requirement of complete zippering for fusion (Song et al., 2021), but it has been unclear whether this activity of Sec17 and Sec18 is directly coupled to HOPS. HOPS can be replaced for fusion by a synthetic tether when the three Q-SNAREs are pre-assembled. We now report that fusion intermediates with arrested SNARE zippering, formed with a synthetic tether but without HOPS, support Sec17/Sec18-triggered fusion. This zippering-bypass fusion is thus a direct result of Sec17 and Sec18 interactions: with each other, with the platform of partially zippered SNAREs, and with the apposed tethered membranes. As these fusion elements are shared among all exocytic and endocytic traffic, Sec17 and Sec18 may have a general role in directly promoting fusion.

Membrane Fusion can be Driven by Sec18/NSF, Sec17/αSNAP, and trans-SNARE complex without HOPS

Yeast vacuolar membrane fusion has been reconstituted with R, Qa, Qb, and Qc-family SNAREs, Sec17/αSNAP, Sec18/NSF, and the hexameric HOPS complex. HOPS tethers membranes and catalyzes SNARE assembly into RQaQbQc trans-complexes which zipper through their SNARE domains to promote fusion. Previously, we demonstrated that Sec17 and Sec18 can bypass the requirement of complete zippering for fusion (Song et al., 2021), but it has been unclear whether this activity of Sec17 and Sec18 is directly coupled to HOPS. HOPS can be replaced for fusion by a synthetic tether when the three Q-SNAREs are pre-assembled. We now report that SNARE zippering-arrested fusion intermediates that are formed without HOPS support Sec17/Sec18-triggered fusion. This zippering-bypass fusion is thus a direct result of Sec17 and Sec18 interactions: with each other, with the platform of partially zippered SNAREs, and with the apposed tethered membranes. As these fusion elements are shared among all exocytic and endocytic traffic, Sec17 and Sec18 may have a general role in directly promoting fusion.

Lipid Droplets and Their Autophagic Turnover via the Raft-Like Vacuolar Microdomains

Although once perceived as inert structures that merely serve for lipid storage, lipid droplets (LDs) have proven to be the dynamic organelles that hold many cellular functions. The LDs’ basic structure of a hydrophobic core consisting of neutral lipids and enclosed in a phospholipid monolayer allows for quick lipid accessibility for intracellular energy and membrane production. Whereas formed at the peripheral and perinuclear endoplasmic reticulum, LDs are degraded either in the cytosol by lipolysis or in the vacuoles/lysosomes by autophagy. Autophagy is a regulated breakdown of dysfunctional, damaged, or surplus cellular components. The selective autophagy of LDs is called lipophagy. Here, we review LDs and their degradation by lipophagy in yeast, which proceeds via the micrometer-scale raft-like lipid domains in the vacuolar membrane. These vacuolar microdomains form during nutrient deprivation and facilitate internalization of LDs via the vacuolar membrane invagination and scission. The resultant intra-vacuolar autophagic bodies with LDs inside are broken down by vacuolar lipases and proteases. This type of lipophagy is called microlipophagy as it resembles microautophagy, the type of autophagy when substrates are sequestered right at the surface of a lytic compartment. Yeast microlipophagy via the raft-like vacuolar microdomains is a great model system to study the role of lipid domains in microautophagic pathways.