24 Jan 2023
by Richa

Summary- paper 11: MitoStores: chaperone-controlled protein granules store mitochondrial precursors in the cytosol

Lena Krämer, Niko Dalheimer, Markus Räschle, Zuzana Storchová, Jan Pielage, Felix Boos, Johannes M Herrmann

The EMBO Journal, 2023

Questions/gaps addressed:

  • Most mitochondrial proteins are synthesized and folded in the cytosol, then imported into mitochondria by translocases in the mito membranes. Accumulation of nonimported precursor proteins in the cytosol upregulates Rpn4 (transcription factor)-dependent Unfolded Protein Response activated by mistargeting of proteins (UPRam). How does the cell handle the stress of mito protein accumulation in the cytosol when UPRam upregulation is blocked?

Key methods:

  • Overexpression cloggers- mito precursors (b2Δ-DHFR or b2-DHFR) and monitor effect of Rpn4 deletion on growth.

  • Comparison of total cellular proteomes of wild-type and rpn4∆ cells with/without clogger induction by TMT MS.

  • Affinity purification with nanotraps-MS from yeast lysates (+/- clogger) to identify the content of Hsp104-GFP-bound structures.

  • Split-GFP approach (first 10 beta sheets of superfolder GFP in mitochondria (Oxa1-GFP1-10)) and strand 11 of GFP on candidates to confirm location.

Major takeaways:

  • Overexpression of slowly imported mito precursors (b2Δ-DHFR or b2-DHFR) called cloggers, causes growth arrest. OE in rpn4∆ mutant no longer arrested for growth. Rpn4 dependent toxicity was specific for mito precursors, no effect on OE of cytosolic aggregation-prone proteins eg., polyglutamine protein Q97-GFP, luciferase(R188Q R261Q), or Ubc9 Y68L.

  • OE of clogger in rpn4∆ cells: proteasome subunits downregulated, cytosolic chaperone Hsp104 (disaggregase) and Hsp42 (small heat shock protein) upregulated. rpn4∆ hsp42∆ hsp104∆ triple mutant cannot grow on respiratory media/nonfermentable carbon sources.

  • Hsp104-GFP accumulates in cytosolic puncta upon clogger OE. IP-MS of Hsp104-GFP structures from cells identified multiple mitochondrial proteins in the IP. Authors call these structure ‘MitoStores’. Found Pdb1, Atp14, Tdh2, Cox8, Cox13, Mrp3 as stronger Hsp104 interactors in rpn4∆ bkg.