The entry of unclosed autophagosomes into vacuoles and its physiological relevance.

It is widely stated in the literature that closed mature autophagosomes (APs) fuse with lysosomes/vacuoles during macroautophagy/autophagy. Previously, we showed that unclosed APs accumulated as clusters outside vacuoles in Vps21/Rab5 and ESCRT mutants after a short period of nitrogen starvation. However, the fate of such unclosed APs remains unclear. In this study, we used a combination of cellular and biochemical approaches to show that unclosed double-membrane APs entered vacuoles and formed unclosed single-membrane autophagic bodies after prolonged nitrogen starvation or rapamycin treatment. Vacuolar hydrolases, vacuolar transport chaperon (VTC) proteins, Ypt7, and Vam3 were all involved in the entry of unclosed double-membrane APs into vacuoles in Vps21-mutant cells. Overexpression of the vacuolar hydrolases, Pep4 or Prb1, or depletion of most VTC proteins promoted the entry of unclosed APs into vacuoles in Vps21-mutant cells, whereas depletion of Pep4 and/or Prb1 delayed the entry into vacuoles. In contrast to the complete infertility of diploid cells of typical autophagy mutants, diploid cells of Vps21 mutant progressed through meiosis to sporulation, benefiting from the entry of unclosed APs into vacuoles after prolonged nitrogen starvation. Overall, these data represent a new observation that unclosed double-membrane APs can enter vacuoles after prolonged autophagy induction, most likely as a survival strategy.

The ESCRT-III complex contributes to macromitophagy in yeast.

Mitochondria play important roles in energy generation and homeostasis maintenance in eukaryotic cells. The damaged or superfluous mitochondria can be nonselectively or selectively removed through the autophagy/lysosome pathway, which was referred as mitophagy. According to the molecular machinery for degrading mitochondria, the selectively removed mitochondria can occur through macromitophagy or micromitophagy. In this study, we show that the endosomal sorting complex required for transport III (ESCRT-III) in budding yeast regulates macromitophagy induced by nitrogen starvation, but not by the post-logarithmic phase growth in lactate medium by monitoring a mitochondrial marker, Om45. Firstly, loss of ESCRT-III subunit Snf7 or Vps4-Vta1 complex subunit Vps4, two representative subunits of the ESCRT complex, suppresses the delivery and degradation of Om45-GFP to vacuoles. Secondly, we show that the mitochondrial marker Om45 and mitophagy receptor Atg32 accumulate on autophagosomes marked with Atg8 (mitophagosomes, MPs) in ESCRT mutants. Moreover, the protease-protection assay indicates that Snf7 and Vps4 are involved in MP closure. Finally, Snf7 interacts with Atg11, which was detected by two ways, glutathione-S-transferase (GST) pulldown and bimolecular fluorescence complementation (BiFC) assay, and this BiFC interaction happens on mitochondrial reticulum. Therefore, we proposed that the ESCRT-III machinery mediates nitrogen starvation-induced macromitophagy by the interaction between Snf7 and Atg11 so that Snf7 is recruited to Atg32-marked MPs by the known Atg11-Atg32 interaction to seal them. These results reveal that the ESCRT-III complex plays a new role in yeast on macromitophagy.

Yeast phospholipase D, Spo14, is not required for macroautophagy.

Autophagy-related gene (Atg) proteins are key players in autophagy. Some proteins that function in vesicle trafficking and lipid metabolism are also involved in autophagy. The SPO14 in yeast, which encodes phospholipase D (PLD), is involved in membrane trafficking and plays a vital role in sporulation during meiosis. Crosstalk has been identified between autophagy and sporulation. Although the PLD is required for macroautophagy in mammals, its role in yeast macroautophagy remains unclear. We observed that Spo14 is not required for macroautophagy in yeast and that it is dispensable for Atg8 lipidation, which plays an important role in phagophore extension. Our results also revealed that green fluorescent protein (GFP)-Atg8 degradation is not completely blocked in atg1Δ/atg1Δ cells under sporulation condition. Therefore, Spo14 is not required for macroautophagy in yeast.

Vps21 Directs the PI3K-PI(3)P-Atg21-Atg16 Module to Phagophores via Vps8 for Autophagy.

Phosphatidylinositol 3-phosphate (PI(3)P) serves important functions in endocytosis, phagocytosis, and autophagy. PI(3)P is generated by Vps34 of the class III phosphatidylinositol 3-kinase (PI3K) complex. The Vps34-PI3K complex can be divided into Vps34-PI3K class II (containing Vps38, endosomal) and Vps34-PI3K class I (containing Atg14, autophagosomal). Most PI(3)Ps are associated with endosomal membranes. In yeast, the endosomal localization of Vps34 and PI(3)P is tightly regulated by Vps21-module proteins. At yeast phagophore assembly site (PAS) or mammalian omegasomes, PI(3)P binds to WD-repeat protein interacting with phosphoinositide (WIPI) proteins to further recruit two conjugation systems, Atg5-Atg12·Atg16 and Atg8-PE (LC3-II), to initiate autophagy. However, the spatiotemporal regulation of PI(3)P during autophagy remains obscure. Therefore, in this study, we determined the effect of Vps21 on localization and interactions of Vps8, Vps34, Atg21, Atg8, and Atg16 upon autophagy induction. The results showed that Vps21 was required for successive colocalizations and interactions of Vps8-Vps34 and Vps34-Atg21 on endosomes, and Atg21-Atg8/Atg16 on the PAS. In addition to disrupted localization of the PI3K complex II subunits Vps34 and Vps38 on endosomes, the localization of the PI3K complex I subunits Vps34 and Atg14, as well as Atg21, was partly disrupted from the PAS in vps21∆ cells. The impaired PI3K-PI(3)P-Atg21-Atg16 axis in vps21∆ cells might delay autophagy, which is consistent with the delay of early autophagy when Atg21 was absent. This study provides the first insight into the upstream sequential regulation of the PI3K-PI(3)P-Atg21-Atg16 module by Vps21 in autophagy.

Genome-wide screening of budding yeast with honokiol to associate mitochondrial function with lipid metabolism.

Honokiol (HNK), an important medicinal component of Magnolia officinalis, is reported to possess pharmacological activities against a variety of diseases. However, the molecular mechanisms of HNK medicinal functions are not fully clear. To systematically study the mechanisms of HNK action, we screened a yeast mutant library based on the conserved nature of its genes among eukaryotes. We identified genes associated with increased resistance or sensitivity to HNK after mutation. After functional classification of these genes, we found that most HNK-resistant strains in the largest functional category were petites with mutations in mitochondrial genes, indicating that mitochondria were related to HNK resistance. Additional analysis showed that resistance of petite mutants to HNK was associated with upregulation of the ATP-binding cassette transporter Pdr5, which pumps out HNK. We also found that several HNK-sensitive mitochondria mutants were not petites, and had larger lipid droplets (LDs). Furthermore, HNK treatment on wild-type yeast cells seemed to disrupt mitochondrial morphology, induced triacylglycerol synthesis, and generated supersized LDs surrounded by mitochondria and endoplasmic reticulum (ER). These changes are also applied to atp7Δ mutant if no carbon resource was available. These results suggested that HNK treatment partly impaired normal mitochondrial function to form larger LDs by altering lipid metabolism.

A Rab5 GTPase module is important for autophagosome closure.

In the conserved autophagy pathway, the double-membrane autophagosome (AP) engulfs cellular components to be delivered for degradation in the lysosome. While only sealed AP can productively fuse with the lysosome, the molecular mechanism of AP closure is currently unknown. Rab GTPases, which regulate all intracellular trafficking pathways in eukaryotes, also regulate autophagy. Rabs function in GTPase modules together with their activators and downstream effectors. In yeast, an autophagy-specific Ypt1 GTPase module, together with a set of autophagy-related proteins (Atgs) and a phosphatidylinositol-3-phosphate (PI3P) kinase, regulates AP formation. Fusion of APs and endosomes with the vacuole (the yeast lysosome) requires the Ypt7 GTPase module. We have previously shown that the Rab5-related Vps21, within its endocytic GTPase module, regulates autophagy. However, it was not clear which autophagy step it regulates. Here, we show that this module, which includes the Vps9 activator, the Rab5-related Vps21, the CORVET tethering complex, and the Pep12 SNARE, functions after AP expansion and before AP closure. Whereas APs are not formed in mutant cells depleted for Atgs, sealed APs accumulate in cells depleted for the Ypt7 GTPase module members. Importantly, depletion of individual members of the Vps21 module results in a novel phenotype: accumulation of unsealed APs. In addition, we show that Vps21-regulated AP closure precedes another AP maturation step, the previously reported PI3P phosphatase-dependent Atg dissociation. Our results delineate three successive steps in the autophagy pathway regulated by Rabs, Ypt1, Vps21 and Ypt7, and provide the first insight into the upstream regulation of AP closure.

Vrl1 relies on its VPS9-domain to play a role in autophagy in Saccharomyces cerevisiae.

Autophagy is an intracellular degradation process involving many Atg proteins, which are recruited hierarchically to regulate this process. Rab/Ypt GTPases and their activators, guanine nucleotide exchange factors (GEFs), which are critical for regulating vesicle trafficking, are also involved in autophagy. Previously, we reported that yeast Vps21 and its GEF Vps9 are required for autophagy. Later, a third yeast VPS9-domain-containing protein, VARP-like 1 (Vrl1), which was identified as a mutant in major laboratory strains, had partially overlapping functions with Vps9 in trafficking. In this study, we showed that Vrl1 performed roles in autophagy, and its VPS9-domain was crucial for its role in autophagy. We found that localization of Vrl1 differed from the other two VPS9-domain-containing proteins, Vps9 and Muk1, and only Vrl1 changed from multipoint to diffusion after starvation. Like Vps9, Vrl1 suppressed autophagic defects caused by the VPS9 deletion. We further showed that these VPS9-domain-containing proteins, Vps9, Muk1, and Vrl1, all co-localized with Atg8 on autophagosomes in cells blocked in any late step of starvation-induced autophagy, with Vrl1 most often co-localizing with Atg8. A small portion (<25%) of these VPS9-domain-containing proteins were degraded through autophagy. However, a large portion (>60%) of Vrl1 decreased independently of autophagy. We propose that Vrl1 may regulate autophagy in a similar way as Vps9, and the level of Vrl1 partly decreases through both autophagy-dependent and -independent routes.

Polymer Additives with Gas Barrier and Anti-Aging Properties Made from Asphaltenes via Supercritical Ethanol.

Asphaltene is often regarded as an undesirable by-product of petroleum processing, possesses vast reserves with little market value. The typical routes of consuming asphaltene, namely burning and landfilling, pose significant environmental challenges. In this study, low-value asphaltene is converted into high-value ethylated carbon clusters (ECC) using a supercritical ethanol technique. The resulting ECC powder demonstrates promising properties for high density polyethylene (HDPE) composite applications. The effects of incorporating ECC on the mechanical, gas barrier, and anti-aging properties of the composite are investigated. Results show that a 1 wt.% ECC led to a 4.2% and 43.5% increase in tensile strength and elongation at break, a reduction of 45.8% and 30.7% in oxygen and carbon dioxide permeability. Furthermore, ECC exhibits effective UV spectrum absorption and conversion in the wavelength range of 400-600 nm, providing protection against UV spectrum damage to HDPE. The incorporation of ECC not only enhances the properties of polymer composites but also sequesters carbon within the polymer matrix, enabling the valorization of asphaltene while mitigating environmental impact.

Autophagosome closure by ESCRT: Vps21/RAB5-regulated ESCRT recruitment via an Atg17-Snf7 interaction.

The macroautophagy/autophagy pathway includes successive steps of phagophore assembly structure formation, phagophore expansion, autophagosome (AP) closure and AP fusion with the lysosome/vacuole. Although information about regulators, factors and molecular mechanisms important for early and late steps of autophagy is abundant, information about AP closure is scarce. In 2017, we reported that the Vps21/RAB5 GTPase module regulates AP closure in yeast. In a recent paper, we show that Vps21 regulates the recruitment of ESCRT to APs to catalyze their closure by controlling an Atg17-Snf7 interaction. Thus, we identify a regulator, a factor, and a molecular mechanism important for AP closure. Abbreviations: AP: Autohagosome; Atg: autophagy-related gene; ESCRT: the endosomal sorting complex required for transport; ILVs: intralumenal vescicles.

Rab5-dependent autophagosome closure by ESCRT.

In the conserved autophagy pathway, autophagosomes (APs) engulf cellular components and deliver them to the lysosome for degradation. Before fusing with the lysosome, APs have to close via an unknown mechanism. We have previously shown that the endocytic Rab5-GTPase regulates AP closure. Therefore, we asked whether ESCRT, which catalyzes scission of vesicles into late endosomes, mediates the topologically similar process of AP sealing. Here, we show that depletion of representative subunits from all ESCRT complexes causes late autophagy defects and accumulation of APs. Focusing on two subunits, we show that Snf7 and the Vps4 ATPase localize to APs and their depletion results in accumulation of open APs. Moreover, Snf7 and Vps4 proteins complement their corresponding mutant defects in vivo and in vitro. Finally, a Rab5-controlled Atg17-Snf7 interaction is important for Snf7 localization to APs. Thus, we unravel a mechanism in which a Rab5-dependent Atg17-Snf7 interaction leads to recruitment of ESCRT to open APs where ESCRT catalyzes AP closure.

[The submental artery perforator flap for lower face defect and deformity].

OBJECTIVE: To analyze the clinical application of submental artery perforator flap for lower face defect and deformity. METHODS: From Sep. 2006 to Mar. 2009, 22 cases with lower face defects and deformities were treated with the submental artery perforator flaps. The age of the patients ranged from 14 to 36. The perforator point was detected by Doppler flowmeter. The size of the flaps ranged from 4 cm x 5 cm to 6 cm x 7 cm. RESULTS: Distal partial necrosis happened in one flap, which healed through dressing. All the other flaps survived with satisfactory appearance and less morbidity in donor sites. CONCLUSIONS: Submental artery perforator flap is very suitable for lower face defect and deformity with reliable blood supply and less morbidity in donor site.

[Superior or inferior gluteal artery perforator flaps for the gluteal sores].

OBJECTIVE: To investigate the clinical application of superior or inferior gluteal artery perforator flaps for the gluteal sores. METHODS: Before operation, the perforator artery was detected by Doppler flowmeter and labeled. The perforator flap was designed, including the perforator artery, but not the gluteal maximum muscle. RESULTS: From Aug. 2006 to May 2009, 15 cases were treated. The flap size ranged from 6 cm x 8 cm to 7 cm x 15 cm. All the flaps survived completely without hematoma, seroma or other complication. CONCLUSIONS: The gluteal maximum muscle-reserved gluteal artery perforator flap is a good choice for gluteal sore with reliable blood supply and less morbidity in donor site.

[Clinical application of thoracodorsal artery perforator flaps].

OBJECTIVE: To analyses the clinical application of thoracodorsal artery perforator flaps (TAP). METHODS: We used free or pedicled TAP flaps in 7 patients from Aug 2006 to April 2007, The age ranged from 7 to 42 years old, the perforator arteries was detected and labeled with a hand held Doppler flowmeter, the size of flaps ranged from 6 cm x 9 cm - 12 cm x 16 cm, the flaps designed with perforator artery included, all the flaps are based on the first perforator artery. RESULTS: All the flaps survived well, no complication occurred with lowest donor site morbidity. CONCLUSIONS: The thoracodorsal artery flap with latissimus dorsal muscle saved is a thin and reliable flaps with robust of blood supply, the flap can reduce significantly donor site morbidity and is a good choice for reconstructive surgery.