Summary-Paper 26

15 Mar 2023
by Richa
Share
tweet

Summary- paper 26: Yeast Svf1 binds ceramides and contributes to sphingolipid metabolism at the ER cis-Golgi interface

Sergej Limar, Carolin Körner, Fernando Martínez-Montañés, Viktoriya G. Stancheva, Verena N. Wolf, Stefan Walter, Elizabeth A. Miller, Christer S. Ejsing, Vanesa Viviana Galassi, Florian Fröhlich

Journal of Cell Biology, 2023

Questions/gaps addressed:

  • Ceramides are synthesized in the ER and receive their head-groups in the Golgi apparatus, yielding complex sphingolipids (such as sphingomyelin in mammalian cells or inositol-containing ceramides in yeast). Transport of ceramides between the ER and the Golgi is performed by the ceramide transport protein (CERT) in mammalian cells. CERT is not conserved in yeast, how is ceramide transport accomplished in yeast?

  • Several candidates for ceramide transport in yeast have been proposed: non-vesicular: Nvj2, Tricalbins, OSBP homologs Osh2, Osh3, and Osh4, and vesicular trafficking. Exact mechanism and contributions unclear.

Major hypotheses:

  • Since Nvj2-mediated ceramide transport occurs only under conditions of high ER stress, hypothesized that other transporter proteins are likely involved.

Key methods:

  • Analyzed high-throughput chemical genetics (Fitdb) dataset to identify genetic similarity to Nvj2. Found Svf1 shows high correlation with Nvj2, Osh1, Osh3.

  • Tetrad analysis and negative genetic interaction: Between svf1∆ and osh2Δ osh3Δ osh4Δ; and nvj2∆ and osh2Δosh3Δosh4Δ. svf1∆ nvj2∆ sensitive to myriocin (0.5 µM).

  • Fluorescence microscopy and localization of C-term Svf1-GFP (colocalized with cis-Golgi marker Mnn9-mKate, and cytoplasm). N-term tagged GFP-Svf1 is not functional.

  • HeliQuest analysis to identify N-terminal membrane targeting motif. Found a potential amphipathic helix (with one face made of hydrophobic residues and the other one containing polar residues).

  • Mutating the hydrophobic valine (position 12) to negatively charged aspartic acid, largely destroying the hydrophobic moment, led to loss of cis-Golgi puncta. The mutant also appears to be less stable, and unable to complement function in svf1∆ sur2∆ mutant.

  • Tested requirement of N-terminal acetylation for membrane insertion (similar to that reported for Grh1) by mutating the second aa (L2E), or using a mutant lacking MAK3, the catalytic subunit of NatC acetyl transferase. Used mass spec of the N-terminus of Svf1 from Svf1-GFP pull down to monitor N-terminally acetylation.

  • Minimal fusion constructs of the first 18 aa of Svf1 tagged with GFP localize to the ER (no cis-Golgi). Addition of amphipathic helix of Grh1 does not recue Golgi localization of function.

  • SILAC MS to identify interactors in Svf1-GFP immunoprecipitates. Found several ER and early-Golgi membrane proteins.

  • Tested the “hug-and-kiss” model for Svf1 at cis-Golgi and ER exit sites contact at the same time- ∼40% of Svf1 dots co-localized with both Mnn9-Kate (cis-Golgi) and Sec31-Halo (ER-exit sites).

  • Tested whether COP-II is required for Svf1 targeting to the Golgi, Svf1-GFP in temperature-sensitive alleles of Sec23 (Sar1 GTPase-activating protein) or Sec17 (α-SNAP co-chaperone). Saw a reduction with cis-Golgi, but inconclusive.

  • Measured the levels of yeast sphingolipids using LC-MS/MS. Also measured content of ceramides on in vitro budded COP-II vesicles. Saw decrease in complex sphingolipids, observed additive effects when combined with sec12 ts (COPII) mutant.

  • Deletion of Svf1 in mutants lacking diacylglycerol acyltransferases (dga1Δ lro1Δ are1Δ are2Δ)- needed for detoxification of ceramides, led to high sensitivity to IPC synthase inhibitor Auroebasidin A (AbA) (added to further increase ceramide toxicity).

  • AlphaFold structural prediction of Svf1 predicts two lipocalin domains made of anti-parallel β-sheets. Used AutoDock Vina molecular docking to predict ceramide binding sites. Two histidine residues located in a small α-helical stretch that lies over the potential ceramide binding pocket. Also mutated to alanines (H273A/H274A). The mutant lost ceramide binding and Golgi localization, and shows genetic interaction with sur2∆.

  • Tested binding to ceramides, immunoprecipitated overexpressed (from pGal promoter) FLAG tagged Svf1, Svf1V12D, and Svf1G7A/G8A without detergents. Extracted lipids from the immunoprecipitates, determined the amount of co-purified ceramide 44:0;4 and, as a control, 34:1 phosphatidylcholine (PC 34:1) using targeted lipidomics. Observed enrichment of 44:0;4 ceramide for all WT and mutants- so amphipathic helix not important for ceramide binding.

Major takeaways:

  • Identified the yeast protein Svf1 is a novel ceramide-binding protein. Svf1 deletion leads to accumulation of ceramides and decrease in complex sphingolipids. Show Svf1 binds ceramides, but how is the binding and release cycle regulated?

  • Also possible that Svf1 does not directly transport ceramides but acts as a sensor for ceramides which then regulate downstream Aur1 activity in the Golgi apparatus.