UVRAG cooperates with cargo receptors to assemble the ER-phagy site.

ER-phagy is a selective autophagy process that targets specific regions of the endoplasmic reticulum (ER) for removal via lysosomal degradation. During cellular stress induced by starvation, cargo receptors concentrate at distinct ER-phagy sites (ERPHS) to recruit core autophagy proteins and initiate ER-phagy. However, the molecular mechanism responsible for ERPHS formation remains unclear. In our study, we discovered that the autophagy regulator UV radiation Resistance-Associated Gene (UVRAG) plays a crucial role in orchestrating the assembly of ERPHS. Upon starvation, UVRAG localizes to ERPHS and interacts with specific ER-phagy cargo receptors, such as FAM134B, ATL3, and RTN3L. UVRAG regulates the oligomerization of cargo receptors and facilitates the recruitment of Atg8 family proteins. Consequently, UVRAG promotes efficient ERPHS assembly and turnover of both ER sheets and tubules. Importantly, UVRAG-mediated ER-phagy contributes to the clearance of pathogenic proinsulin aggregates. Remarkably, the involvement of UVRAG in ER-phagy initiation is independent of its canonical function as a subunit of class III phosphatidylinositol 3-kinase complex II.

TOR-mediated Ypt1 phosphorylation regulates autophagy initiation complex assembly.

The regulation of autophagy initiation is a key step in autophagosome biogenesis. However, our understanding of the molecular mechanisms underlying the stepwise assembly of ATG proteins during this process remains incomplete. The Rab GTPase Ypt1/Rab1 is recognized as an essential autophagy regulator. Here, we identify Atg23 and Atg17 as binding partners of Ypt1, with their direct interaction proving crucial for the stepwise assembly of autophagy initiation complexes. Disruption of Ypt1-Atg23 binding results in significantly reduced Atg9 interactions with Atg11, Atg13, and Atg17, thus preventing the recruitment of Atg9 vesicles to the phagophore assembly site (PAS). Likewise, Ypt1-Atg17 binding contributes to the PAS recruitment of Ypt1 and Atg1. Importantly, we found that Ypt1 is phosphorylated by TOR at the Ser174 residue. Converting this residue to alanine blocks Ypt1 phosphorylation by TOR and enhances autophagy. Conversely, the Ypt1S174D phosphorylation mimic impairs both PAS recruitment and activation of Atg1, thus inhibiting subsequent autophagy. Thus, we propose TOR-mediated Ypt1 as a multifunctional assembly factor that controls autophagy initiation via its regulation of the stepwise assembly of ATG proteins.

Vps13 is required for the packaging of the ER into autophagosomes during ER-phagy.

Endoplasmic reticulum (ER) macroautophagy (hereafter called ER-phagy) uses autophagy receptors to selectively degrade ER domains in response to starvation or the accumulation of aggregation-prone proteins. Autophagy receptors package the ER into autophagosomes by binding to the ubiquitin-like yeast protein Atg8 (LC3 in mammals), which is needed for autophagosome formation. In budding yeast, cortical and cytoplasmic ER-phagy requires the autophagy receptor Atg40. While different ER autophagy receptors have been identified, little is known about other components of the ER-phagy machinery. In an effort to identify these components, we screened the genome-wide library of viable yeast deletion mutants for defects in the degradation of cortical ER following treatment with rapamycin, a drug that mimics starvation. Among the mutants we identified was vps13Δ. While yeast has one gene that encodes the phospholipid transporter VPS13, humans have four vacuolar protein-sorting (VPS) protein 13 isoforms. Mutations in all four human isoforms have been linked to different neurological disorders, including Parkinson's disease. Our findings have shown that Vps13 acts after Atg40 engages the autophagy machinery. Vps13 resides at contact sites between the ER and several organelles, including late endosomes. In the absence of Vps13, the cortical ER marker Rtn1 accumulated at late endosomes, and a dramatic decrease in ER packaging into autophagosomes was observed. Together, these studies suggest a role for Vps13 in the sequestration of the ER into autophagosomes at late endosomes. These observations may have important implications for understanding Parkinson's and other neurological diseases.

ER-phagy requires Lnp1, a protein that stabilizes rearrangements of the ER network.

The endoplasmic reticulum (ER) forms a contiguous network of tubules and sheets that is predominantly associated with the cell cortex in yeast. Upon treatment with rapamycin, the ER undergoes degradation by selective autophagy. This process, termed ER-phagy, requires Atg40, a selective autophagy receptor that localizes to the cortical ER. Here we report that ER-phagy also requires Lnp1, an ER membrane protein that normally resides at the three-way junctions of the ER network, where it serves to stabilize the network as it is continually remodeled. Rapamycin treatment increases the expression of Atg40, driving ER domains marked by Atg40 puncta to associate with Atg11, a scaffold protein needed to form autophagosomes. Although Atg40 largely localizes to the cortical ER, the autophagy machinery resides in the cell interior. The localization of Atg40 to sites of autophagosome formation is blocked in an lnp1Δ mutant or upon treatment of wild-type cells with the actin-depolymerizing drug Latrunculin A. This prevents the association of Atg40 with Atg11 and the packaging of the ER into autophagosomes. We propose that Lnp1 is needed to stabilize the actin-dependent remodeling of the ER that is essential for ER-phagy.

A novel Drosophila mitochondrial carrier protein acts as a Mg(2+) exporter in fine-tuning mitochondrial Mg(2+) homeostasis.

The homeostasis of magnesium (Mg(2+)), an abundant divalent cation indispensable for many biological processes including mitochondrial functions, is underexplored. In yeast, the mitochondrial Mg(2+) homeostasis is accurately controlled through the combined effects of importers, Mrs2 and Lpe10, and an exporter, Mme1. However, little is known about this Mg(2+) homeostatic process in multicellular organisms. Here, we identified the first mitochondrial Mg(2+) transporter in Drosophila, the orthologue of yeast Mme1, dMme1, by homologous comparison and functional complementation. dMme1 can mediate the exportation of mitochondrial Mg(2+) when heterologously expressed in yeast. Altering the expression of dMme1, although only resulting in about a 10% change in mitochondrial Mg(2+) levels in either direction, led to a significant survival reduction in Drosophila. Furthermore, the reduced survival resulting from dMme1 expression changes could be completely rescued by feeding the dMME1-RNAi flies Mg(2+)-restricted food or the dMME1-over-expressing flies the Mg(2+)-supplemented diet. Our studies therefore identified the first Drosophila mitochondrial Mg(2+) exporter, which is involved in the precise control of mitochondrial Mg(2+) homeostasis to ensure an optimal state for survival.

A novel mitochondrial carrier protein Mme1 acts as a yeast mitochondrial magnesium exporter.

The homeostasis of magnesium (Mg2+), an abundant divalent cation indispensable for many biological processes including mitochondrial functions, is underexplored. Previously, two mitochondrial Mg2+ importers, Mrs2 and Lpe10, were characterized for mitochondrial Mg2+ uptake. We now show that mitochondrial Mg2+ homeostasis is accurately controlled through the combined effects of previously known importers and a novel exporter, Mme1 (mitochondrial magnesium exporter 1). Mme1 belongs to the mitochondrial carrier family and was isolated for its mutation that is able to suppress the mrs2Δ respiration defect. Deletion of MME1 significantly increased steady-state mitochondrial Mg2+ concentration, while overexpression decreased it. Measurements of Mg2+ exit from proteoliposomes reconstituted with purified Mme1 provided definite evidence for Mme1 as an Mg2+ exporter. Our studies identified, for the first time, a mitochondrial Mg2+ exporter that works together with mitochondrial importers to ensure the precise control of mitochondrial Mg2+ homeostasis.

Enhancing neuronal reticulophagy: a strategy for combating aging and APP toxicity.

Reticulophagy, which directs the endoplasmic reticulum (ER) to the phagophore for sequestration within an autophagosome and subsequent lysosomal degradation via specific receptors, is essential for ER quality control and is implicated in various diseases. This study utilizes Drosophila to establish an in vivo model for reticulophagy. Starvation-induced reticulophagy is detected across multiple tissues in Drosophila. Whole-body upregulation or downregulation of the expression of reticulophagy receptors, atl and Rtnl1, negatively affects fly health. Notably, moderate upregulation of reticulophagy in neuronal tissues by overexpressing these receptors reduces age-related degeneration. In a Drosophila Alzheimer model expressing human APP (amyloid beta precursor protein), reticulophagy is compromised. Correcting reticulophagy by enhancing atl and Rtnl1 expression in the neurons promotes APP degradation, significantly reducing neurodegenerative symptoms. However, overexpression of mutated atl and Rtnl1, which disrupts the interaction of the corresponding proteins with Atg8, does not alleviate these symptoms, emphasizing the importance of receptor functionality. These findings support modulating reticulophagy as a therapeutic strategy for aging and neurodegenerative diseases associated with ER protein accumulation.

UVRAG: orchestrating the initiation of reticulophagy.

Reticulophagy is a selective autophagy of the endoplasmic reticulum (ER) mediated by cargo receptors. It plays a crucial role in ER quality control, yet the mechanisms that initiate reticulophagy remain poorly understood. Our study identified the multifunctional protein UVRAG (UV radiation resistance associated gene) as a novel regulator of reticulophagy. UVRAG interacts with sheet and tubular reticulophagy receptors, regulates the oligomerization of receptors and facilitates their interaction with LC3/GABARAP, critical for ER fragmentation and autophagosome targeting. Remarkably, we found that UVRAG's function in reticulophagy initiation is independent of its traditional role in macroautophagy. Furthermore, UVRAG enhances the degradation of ER-associated mutant proteins linked to diseases like diabetes. Our findings offer insights into the mechanisms of reticulophagy initiation and highlight UVRAG's therapeutic potential in ER-related diseases.

Upregulation of neuronal ER-phagy improves organismal fitness and alleviates APP toxicity.

ER-phagy, a selective autophagy targeting the endoplasmic reticulum (ER) for lysosomal degradation through cargo receptors, plays a critical role in ER quality control and is linked to various diseases. However, its physiological and pathological roles remain largely unclear due to a lack of animal model studies. This study establishes Drosophila as an in vivo ER-phagy model. Starvation triggers ER-phagy across multiple fly tissues. Disturbing ER-phagy by either globally upregulating or downregulating ER-phagy receptors, Atl or Rtnl1, harms the fly. Notably, moderate upregulation of ER-phagy in fly brains by overexpressing Atl or Rtnl1 significantly attenuates age-associated neurodegenerations. Furthermore, in a Drosophila model of Alzheimer's disease expressing human amyloid precursor protein (APP), impaired ER-phagy is observed. Enhancing ER-phagy in the APP-expressing fly brain facilitates APP degradation, significantly alleviating disease symptoms. Therefore, our findings suggest that modulating ER-phagy may offer a therapeutic strategy to treat aging and diseases associated with ER protein aggregation.

Antimicrobial activity of human islet amyloid polypeptides: an insight into amyloid peptides' connection with antimicrobial peptides.

Human islet amyloid polypeptide (hIAPP) shows an antimicrobial activity towards two types of clinically relevant bacteria. The potency of hIAPP varies with its aggregation states. Circular dichroism was employed to determine the interaction between hIAPP and bacteria lipid membrane mimic. The antimicrobial activity of each aggregate species is associated with their ability to induce membrane disruption. Our findings provide new evidence revealing the antimicrobial activity of amyloid peptide, which suggest a possible connection between amyloid peptides and antimicrobial peptides.

Advances in ER-Phagy and Its Diseases Relevance.

As an important form of selective autophagy in cells, ER-phagy (endoplasmic reticulum-selective autophagy), the autophagic degradation of endoplasmic reticulum (ER), degrades ER membranes and proteins to maintain cellular homeostasis. The relationship between ER-phagy and human diseases, including neurodegenerative disorders, cancer, and other metabolic diseases has been unveiled by extensive research in recent years. Starting with the catabolic process of ER-phagy and key mediators in this pathway, this paper reviews the advances in the mechanism of ER-phagy and its diseases relevance. We hope to provide some enlightenment for further study on ER-phagy and the development of novel therapeutic strategies for related diseases.

A new role for a COPII cargo adaptor in autophagy.

Endoplasmic reticulum (ER) homeostasis is maintained by the removal of misfolded ER proteins via different quality control pathways. Aggregation-prone proteins, including certain disease-linked proteins, are resistant to conventional ER degradation pathways and require other disposal mechanisms. Reticulophagy is a disposal pathway that uses resident autophagy receptors. How these receptors, which are dispersed throughout the ER network, target a specific ER domain for degradation is unknown. We recently showed in budding yeast, that ER stress upregulates the reticulophagy receptor, triggering its association with the COPII cargo adaptor complex, Sfb3/Lst1-Sec23 (SEC24C-SEC23 in mammals), to discrete sites on the ER. These domains are packaged into phagophores for degradation to prevent the accumulation of protein aggregates in the ER. This unconventional role for Sfb3/Lst1 is conserved in mammals and is independent of its role as a cargo adaptor on the secretory pathway. Our findings may have important therapeutic implications in protein-aggregation linked neurodegenerative disorders.

A COPII subunit acts with an autophagy receptor to target endoplasmic reticulum for degradation.

The COPII-cargo adaptor complex Lst1-Sec23 selectively sorts proteins into vesicles that bud from the endoplasmic reticulum (ER) and traffic to the Golgi. Improperly folded proteins are prevented from exiting the ER and are degraded. ER-phagy is an autophagic degradation pathway that uses ER-resident receptors. Working in yeast, we found an unexpected role for Lst1-Sec23 in ER-phagy that was independent from its function in secretion. Up-regulation of the stress-inducible ER-phagy receptor Atg40 induced the association of Lst1-Sec23 with Atg40 at distinct ER domains to package ER into autophagosomes. Lst1-mediated ER-phagy played a vital role in maintaining cellular homeostasis by preventing the accumulation of an aggregation-prone protein in the ER. Lst1 function appears to be conserved because its mammalian homolog, SEC24C, was also required for ER-phagy.

Mitochondrial release of the NADH dehydrogenase Ndi1 induces apoptosis in yeast.

Saccharomyces cerevisiae NDI1 codes for the internal mitochondrial ubiquinone oxidoreductase, which transfers electrons from NADH to ubiquinone in the respiratory chain. Previously we found that Ndi1 is a yeast homologue of the protein apoptosis-inducing factor-homologous mitochondrion-associated inducer of death and displays potent proapoptotic activity. Here we show that S. cerevisiae NDI1 is involved in apoptosis induced by various stimuli tested, including H(2)O(2), Mn, and acetate acid, independent of Z-VAD-fmk (a caspase inhibitor) inhibition. Although Ndi1 also participates in respiration, its proapoptotic property is separable from the ubiquinone oxidoreductase activity. During apoptosis, the N-terminal of Ndi1 is cleaved off in the mitochondria, and this activated form then escapes out to execute its apoptotic function. The N-terminal cleavage appears to be essential for the manifestation of the full apoptotic activity, as the uncleaved form of Ndi1 exhibits much less growth-inhibitory activity. Our results thus indicate an important role of Ndi1 in the switch of life and death fates in yeast: during normal growth, Ndi1 assimilates electrons to the electron transport chain and initiates the respiration process to make ATP, whereas under stresses, it cleaves the toxicity-sequestering N-terminal cap, is released from the mitochondria, and becomes a cell killer.