Summary-Paper 50

05 Jun 2023
by Richa
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Summary- paper 50: The wtf4 meiotic driver utilizes controlled protein aggregation to generate selective cell death

Nicole L Nuckolls, Anthony C Mok, Jeffrey J Lange, Kexi Yi, Tejbir S Kandola, Andrew M Hunn, Scott McCroskey, Julia L Snyder, María Angélica Bravo Núñez, Sarah E Zanders

eLife, 2020

Questions/gaps addressed:

  • wtf4 gene is a meiotic driver in Schizosaccharomyces pombe and S kombucha that uses a poison-antidote mechanism to selectively kill meiotic products (spores) that do not inherit wtf4. Spores expressing just the toxin die, while those expressing both the toxin and antidote survive. How do these drivers work and selectively destroy spores?

  • Wtf4 poison and antidote are encoded from overlapping coding sequences, alternate splicing leads to an additional start codon and the antidote contains ~45 additional N-terminal amino acids, the C terminal 293 aa are the same. The two proteins are predicted to contain 6 TMDs.

Key methods:

  • Express S. kombucha wtf4-poison and antidote proteins in S. pombe and in vegetative S. cerevisiae. Inducible over-expression of the poison coding sequence (using the Z3EV β-estradiol inducible system) kills the cells/ spores. Co-expression of the antidote coding sequence restores viability.

  • Fluorescence microscopy, FRET, Distributed Amphifluoric FRET (DAmFRET), half punctum-Fluorescence Recovery After Photobleaching (half-FRAP): show poison localizes in punctate structures, antidote in peri-vacuolar puncta. When co-expressed, both are in the peri-vacuolar structures or inside the vacuole. Poison and the antidote are close enough to physically interact, and likely self-assemble/interact with themselves. When photobleached, do not see recovery up to 3 min, suggesting the assemblies are not very dynamic.

  • Increasing the repeats of 7 amino acid sequence (IGNAFRG) that is found in varying copy numbers in many members of the wtf gene family, in the wtf4 poison or antidote alone, disrupted the interaction, but together restored the neutralization ability.

  • Transmission electron microscopy (TEM): Wtf4 antidote localizes to vesicles adjacent to the vacuole. By fluorescence microscopy that is adjacent to Rnq1 and Atg8 puncta- the authors suggest these to be IPOD structures. They also report accumulation of lipid droplets in cells expressing the toxin-antidote. Also observe mitochondriala hyper-fission.

  • Unbiased SGA screen to identify mutants that don’t grow when the toxin-antidote are expressed- to identify what is the machinery required to neutralize these. Identify several mitochondrial proteins, CWI pathway, ESCRTs, CORVET, Vps1, vacuolar and endosome trafficking machinery. In the absence of many of these, even the antidote by itself is toxic.

Major takeaways:

  • Wtf4 toxin and antidote are membrane proteins, that differ in the N terminal 45 aa. Toxin by itself localizes to dispersed puncta, antidote by itself is peri-vacuolar. Together they are both peri-vacuolar. Why is the toxin toxic and not the antidote? Is it something about the difference in the N term or that the antidote is more effectively cleared due to its peri-vacuolar localization? Why is the localization different- is the N terminus the driver for the localization?

  • Mitochondria, and the ESCRT pathway are two main ways of clearing these toxin-antidote intermediates.