26 Jun 2023
by Richa

Summary- paper 53: The ER calcium channel Csg2 integrates sphingolipid metabolism with autophagy

Shiyan Liu, Mutian Chen, Yichang Wang, Yuqing Lei, Ting Huang, Yabin Zhang, Sin Man Lam, Huihui Li, Shiqian Qi, Jia Geng, Kefeng Lu

Nature communications, 2023

Questions/gaps addressed:

  • Calcium conc in the cytosol is low (0.1 µM), extracellular calcium at 1 mM, intracellular calcium reservoirs (endosome 2 µM, lysosome at 0.5 mM, ER at 0.5 mM, Golgi at 0.1 mM, mitochondria at 0.1 µM and secretory vesicle at 0.1 mM). cytosolic calcium conc is rapidly modulated by ion channels and pumps. What are the consequences of abnormal calcium accumulation in reservoir organelles caused by dysfunctional calcium channels?

  • Cytosolic calcium binds and activates calmodulins that transduce signals into appropriate downstream outputs through calcineurin phosphatase and kinases, regulating pathways such as autophagy. What is the mechanism by which calcium regulates autophagy?

  • Sphingolipids are ubiquitous components of eukaryotic membranes and function as signaling bioactive lipids regulating cell growth, endocytosis, protein trafficking etc. What mechansims regulate sphingolipid homeostasis?

Major hypotheses:

  • Calcium and sphingolipid homeostasis are interlinked and regulate autophagy.

Key methods:

  • Screened calcium channel mutants in yeast (csg2Δ, pmr1Δ, spf1Δ, pmc1Δ, vcx1Δ, yvc1Δ, mid1Δ, rch1Δ, ecm7Δ, cch1Δ) for viability after amino acid starvation.
    • Csg2 was the most defective for growth after 72h in SD-N (mild phenotype for Ecm7, Mid1).
    • Defective for GFP-Atg8, GFP-50Q model reporter processing after 16h in SD-N, 16h in SD-Glu. RFP-Ape1 and Pho8Δ60 failed to be delivered to vacuole lumen for 3h in SD-N.
    • No defect in Ape1 maturation in nutrient rich conditions (although Atg1 is blocked).
    • Pmr1 deletion led to upregulation of autophagy (GFP-Atg8 processing observed within 6h in SD-N). Do transport directions of calcium (into the ER by Pmr1 and out of the ER by Csg2) cause opposite effects on autophagy?
  • To monitor the stage of autophagy that is blocked, looked at GFP-Atg8 localization: diffuse cytosolic distribution of Atg8 indicating the failure of autophagosome fusion with vacuoles. Deletion of the SNARE subunit Vam3 or GTPase Ypt7 lead to accumulation of GFP-Atg8 puncta. No defect in general vacuolar delivery- monitored by CPY-mCh, GFP-Sna3.

  • Csg2 mainly predicted to contain 10 TMDs and a potential calcium binding EF-hand motif. Csg2-GFP, Csg2-HA, and HA-Csg2 complement function. Csg2-GFP localizes to the ER.

  • Purified Csg2 protein, reconstituted into a planar lipid bilayer and measured Ca channel activity using a single-channel conductance assay. Did not observe conductance for Mg, Zn, Mn, Fe ions.

  • A genetic fluorescent probe of ER calcium, Stt3-jGCaMP7c to measure ER Calcium levels and using a kit using ER calcium probe Mag-Fluo-AM. Higher ER calcium levels in Csg2-deleted mutant. jGCaMP7c is a sensitive sensor with low background fluorescence.

  • Functionally important regions: Deletion of any TMD killed function. Deletion of the EF-hand motif had not effect on function or on ER localization.

  • Treatment of csg2∆ cells with 100 mM CaCl2 (up to 800 mM), did not rescue autophagy defect.
    • Treatment with EDTA (5 mM), or EGTA (20 mM), restored autophagy. Increase in ER calcium rather than a decrease responsible for autophagy block in csg2∆.
    • Deleting Csg2 and Pmr1 together (but not vacuolar calcium channels Pmc1, Vcx1, or Yvc1) also restored autophagy.
    • Deletion of the cytosolic calmodulin-calcineurin-Crz1 signaling components Cna1, Cnb1, and Crz1 did not rescue autophagy
  • Comprehensive lipidomics analysis in WT vs csg2∆ under starvation.
    • phytoCers and PHS (immediate precursors of complex sphingolipids) exhibited significant accumulation in the mutant.
    • Complex sphingolipid mannosylinositol phosphorylceramide (MIPC) was reduced in csg2∆.
    • Sphingolipid synthesis pathway genes tested. Tsc10, Lip1, Aur1. No effect of Tsc10, but Lip1 and Aur1 mutants blocked autophagy. Double mutant csg2∆ tsc10-ts restored autophagy. No effect of combining Csg2 and Lip1/Aur1.
    • Steady state stability of Aur1-GFP/ GFP-Aur1 reduced in csg2∆ during starvation. But the levels of Kei1 (in complex with Aur1 for IPS synthesis) not affected.
      • Treatment with EGTA/EDTA rescued Aur1 levels.
      • Aur1 also stable in csg2∆ pmr1∆ double mutant (so dependent on ER calcium levels).
      • Aur1 stable when treated with proteasome inhibitor MG132 but not by the vacuole inhibitor NH4Cl, so degraded by the proteosome.
    • Tor1 activity is higher in csg2∆ during starvation (monitored by Sch9 phosphorylation) also inhibiting autophagy.

Major takeaways:

  • Deletion of Csg2, the ER-resident calcium channel, causes accumulation of calcium in the ER, which disturbs sphingolipid metabolism by destabilizing Aur1 (how?), leading to increases in PHS that block autophagy (how?). Mechanism of PHS regulated autophagy inhibition unclear.