PINK1 controls RTN3L-mediated ER autophagy by regulating peripheral tubule junctions.

Here, we report that the RTN3L-SEC24C endoplasmic reticulum autophagy (ER-phagy) receptor complex, the CUL3KLHL12 E3 ligase that ubiquitinates RTN3L, and the FIP200 autophagy initiating protein, target mutant proinsulin (Akita) condensates for lysosomal delivery at ER tubule junctions. When delivery was blocked, Akita condensates accumulated in the ER. In exploring the role of tubulation in these events, we unexpectedly found that loss of the Parkinson's disease protein, PINK1, reduced peripheral tubule junctions and blocked ER-phagy.

RTN3L and CALCOCO1 function in parallel to maintain proteostasis in the endoplasmic reticulum.

Reticulophagy is mediated by autophagy receptors that function in one of the two domains of the ER, tubules or flat sheets. Three different conserved mammalian receptors mediate autophagy in ER tubules: RTN3L, ATL3 and CALCOCO1. Previous studies have shown that RTN3L maintains proteostasis by targeting mutant aggregation-prone proteins for autophagy at distinct foci in ER tubules that we named ERPHS (ites).

Impairment of transcription factor Gcr1p binding motif perturbs OPI3 transcription in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the transcription factor GCR1 plays a vital role in carbohydrate metabolism and in the current study we tried to elucidate its role in lipid metabolism. In silico analysis revealed the upstream activation sequence (UAS) in the promoter region of OPI3 possessed six conserved recognition sequences for Gcr1p and the ChIP assay confirmed the binding of Gcr1p on the OPI3 promoter region. The real-time quantitative polymerase chain reaction and promoter-reporter activity revealed a substantial reduction in OPI3 expression and was supported with decreased phosphatidylcholine (PC) level that is rescued with exogenous choline supplementation in gcr1∆ cells.

Endoplasmic reticulum tubules limit the size of misfolded protein condensates.

The endoplasmic reticulum (ER) is composed of sheets and tubules. Here we report that the COPII coat subunit, SEC24C, works with the long form of the tubular ER-phagy receptor, RTN3, to target dominant-interfering mutant proinsulin puncta to lysosomes. When the delivery of puncta to lysosomes was disrupted, large puncta accumulated in the ER.

Human OVCA2 and its homolog FSH3-induced apoptosis in Saccharomyces cerevisiae.

Mammalian ovarian tumor suppressor candidate 2 (OVCA2) gene belongs to the family of serine hydrolase (FSH). This study aimed to elucidate the functional similarities of OVCA2 with its yeast homolog genes (FSH1, FSH2, and FSH3) regarding apoptosis. We found that the expression of OVCA2 in Saccharomyces cerevisiae increased production of reactive oxygen species (ROS), decreased cell growth, disturbed mitochondrial morphology, reduced membrane potential, increased chromatin condensation, and externalization of phosphatidylserine (PS) (annexin V/propidium iodide staining) indicating induced apoptotic cell death in yeast.

FSH1 encodes lysophospholipase activity in Saccharomyces cerevisiae.

Objectives: To elucidate the role of FSH1 (family of serine hydrolase) in lipid homeostasis.

Results: Proteins in various species containing alpha/beta hydrolase domain are known to be involved in lipid metabolism. In silico analysis of the FSH1 gene in Saccharomyces cerevisiae revealed the presence of alpha/beta hydrolase domain (ABHD) and a lipase motif (GXSXG), however its function in lipid metabolism remained elusive.

Crosstalk between protein N-glycosylation and lipid metabolism in Saccharomyces cerevisiae.

The endoplasmic reticulum (ER) is a multi functional organelle and plays a crucial role in protein folding and lipid biosynthesis. The SEC59 gene encodes dolichol kinase, required for protein glycosylation in the ER. The mutation of sec59-1 caused a protein N-glycosylation defect mediated ER stress resulting in increased levels of phospholipid, neutral lipid and sterol, whereas growth was reduced.

FSH1 overexpression triggers apoptosis in Saccharomyces cerevisiae.

FSH1 belongs to the family of serine hydrolases in yeast and is homologous to the human ovarian tumor suppressor gene (OVAC2). Our preliminary results showed that cells lacking Fsh1p exhibit an increase in cell growth, and a decrease in the expression of AIF1 and NUC1 (apoptosis responsive genes) when compared to the wild type cells. Growth inhibition of cells overexpressing FSH1 is due to induction of cell death associated with cell death markers typical of mammalian apoptosis namely DNA fragmentation, phosphatidylserine externalization, ROS accumulation, Cytochrome c release, and altered mitochondrial membrane potential.

Impaired GCR1 transcription resulted in defective inositol levels, vacuolar structure and autophagy in Saccharomyces cerevisiae.

In yeast, the GCR1 transcription factor is involved in the regulation of glycolysis and its deletion exhibited growth defect, reduced inositol and phosphatidylinositol (PI) levels compared to WT cells. We observed a down regulation of the INO1 and PIS1 expression in gcr1∆ cells under both I- and I+ conditions and the over expression of GCR1 in gcr1∆ cells restored the growth, retrieved the expression of INO1, and PIS1 comparable to WT cells. In the gel shift assay, the Gcr1p binds to its consensus sequence CTTCC in PIS1 promoter and regulates its expression but not in INO1 transcription.

Endoplasmic reticulum stress and calcium imbalance are involved in cadmium-induced lipid aberrancy in Saccharomyces cerevisiae.

The endoplasmic reticulum is the key organelle which controls protein folding, lipid biogenesis, and calcium (Ca(2+)) homeostasis. Cd exposure in Saccharomyces cerevisiae activated the unfolded protein response and was confirmed by the increased Kar2p expression. Cd exposure in wild-type (WT) cells increased PC levels and the PC biosynthetic genes.

Defect of zinc transporter ZRT1 ameliorates cadmium induced lipid accumulation in Saccharomyces cerevisiae.

Cadmium (Cd) is a non-essential divalent heavy metal that enters the cells by utilizing the transport pathways of the essential metals, like zinc (Zn), in Saccharomyces cerevisiae. This work focuses on Cd accumulation and its impact on deletion of Zn transporters Zrt1p and Zrt2p and lipid homeostasis. Cd exposure reduces the Zn levels in the mutant strains, and the effect was higher in zrt2Δ cells.