Iron Limitation Restores Autophagy and Increases Lifespan in the Yeast Model of Niemann–Pick Type C1

Abstract

Niemann–Pick type C1 (NPC1) is an endolysosomal transmembrane protein involved in the export of cholesterol and sphingolipids to other cellular compartments such as the endoplasmic reticulum and plasma membrane. NPC1 loss of function is the major cause of NPC disease, a rare lysosomal storage disorder characterized by an abnormal accumulation of lipids in the late endosomal/lysosomal network, mitochondrial dysfunction, and impaired autophagy. NPC phenotypes are conserved in yeast lacking Ncr1, an orthologue of human NPC1, leading to premature aging. Herein, we performed a phosphoproteomic analysis to investigate the effect of Ncr1 loss on cellular functions mediated by the yeast lysosome-like vacuoles. Our results revealed changes in vacuolar membrane proteins that are associated mostly with vesicle biology (fusion, transport, organization), autophagy, and ion homeostasis, including iron, manganese, and calcium. Consistently, the cytoplasm to vacuole targeting (Cvt) pathway was increased in ncr1∆ cells and autophagy was compromised despite TORC1 inhibition. Moreover, ncr1∆ cells exhibited iron overload mediated by the low-iron sensing transcription factor Aft1. Iron deprivation restored the autophagic flux of ncr1∆ cells and increased its chronological lifespan and oxidative stress resistance. These results implicate iron overload on autophagy impairment, oxidative stress sensitivity, and cell death in the yeast model of NPC1.

Automated quantification of vacuole fusion and lipophagy in Saccharomyces cerevisiae from fluorescence and cryo-soft X-ray microscopy data using deep learning

“…This could be the direct consequence of impaired delivery of ergosterol into the vacuole membrane in the absence of the Ncr1-Npc2 transport system, in line with our previous results, and the absolute requirement of ergosterol for vacuole fusion [ 8 , 43 , 44 ]. Alternatively, it could be the consequence of other factors, such as a disturbed sphingolipid homeostasis, altered TORC signaling or changed iron availability, which all have been observed in ncr1Δ cells [ 16 , 17 , 45 , 46 ]. To explore such possibilities, we have used inhibitors of the aforementioned processes and assessed their impact on vacuole fusion status in wild-type cells and in cells lacking either Ncr1 or Npc2 under starvation conditions.…”

Sit4 Genetically Interacts with Vps27 to Regulate Mitochondrial Function and Lifespan in Saccharomyces cerevisiae

“…Cells were cultured to the late exponential (Log) phase (OD 600nm ≈ 2) and vacuolar membranes were isolated as described [52,53]. Samples were processed for proteomics analysis and proteins were identified and quantified by mass spectrometry (nanoLC-MS/MS), as described [54,55].…”

“…For the analysis of microautophagy induction, Log phase cells treated for 4 h with 200 ng mL −1 of rapamycin were used as positive control. Proteins were extracted using 0.1 M NaOH (Merck, Darmstadt, Germany), dissolved in Laemmli buffer, and quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA), as described [53]. Protein samples were prepared by adding 5% (v/v) 2-mercaptoethanol (Merck, Darmstadt, Germany), separated by SDS-PAGE, and transferred onto a nitrocellulose membrane (GE Healthcare, Chicago, IL, USA).…”

“…Oxygen consumption rate (OCR) was measured using a Clark-type oxygen electrode coupled to an Oxygraph plus system (Hansatech, Norfolk, UK), as described [53].…”

“…To investigate the impact of Sit4 deficiency on yeast lysosome-like vacuoles, we conducted a proteomic analysis of vacuolar membranes, isolated as described [52,53] from both wild-type and sit4∆ cells grown in SC medium to OD 600nm ≈ 2. We identified a set of 230 vacuolar membrane proteins, which represent approximately 80% of the entire vacuolar membrane proteome of S. cerevisiae.…”