Fur4 mediated uracil-scavenging to screen for surface protein regulators

Abstract

Cell surface membrane proteins perform diverse and critical functions and are spatially and temporally regulated by membrane trafficking pathways. Although perturbations in these pathways underlie many pathologies, our understanding of these pathways at a mechanistic level remains incomplete. Using yeast as a model, we have developed an assay that reports on the surface activity of the Fur4 uracil permease in uracil auxotroph strains grown in the presence of limited uracil. This assay was used to screen a haploid deletion library that identified mutants with both diminished and enhanced comparative growth in restricted uracil media. Factors identified, including various multi-subunit complexes, were enriched for membrane trafficking and transcriptional functions, in addition to various uncharacterised genes. Bioinformatic analysis of expression profiles from many strains lacking identified transcription factors required for efficient uracil-scavenging revealed they control expression of other uracil-scavenging factors, in addition to membrane trafficking genes essential for viability, and therefore not represented in the screen. Finally, we performed a secondary mating factor secretion screen to functionally categorise factors implicated in uracil-scavenging, most of which are conserved throughout evolution.

Investigating molecular crowding during cell division and hyperosmotic stress in budding yeast with FRET

“…To assess if these bud neck crowding events correlate with organelle inheritance, we used time lapse microscopy to follow the inheritance of the vacuole, which is conveniently both a large organelle that is inherited early in the budding process ( Li et al, 2021a ). Using a mNeonGreen tagged version of the uracil permease Fur4, which localises to the plasma membrane and also the vacuolar lumen ( Paine et al, 2021 ), we could track inheritance over time ( Supplemental Video 3 ). We also labelled the vacuole by performing a pulse-chase with media containing the red-fluorescent dye FM4-64 ( Vida & Emr, 1995 ).…”

Recycling of cell surface membrane proteins from yeast endosomes is regulated by ubiquitinated Ist1

“…Tryptophan auxotroph ( trp1∆ ) yeast cells based on the SEY6210 background were grown to mid-log phase before being spotted out across a 10-fold serial dilution and grown on plates of replete (40 mg/liter) and two restricted (5 and 2.5 mg/liter) tryptophan concentrations. To quantify growth, densitometry was used to measure the growth intensity across different dilutions on the plate ( Paine et al, 2021 Preprint ). Yeast growth at each dilution was normalized to a WT control on the same plate, and the difference was plotted for indicated tryptophan concentrations.…”

Recycling of cell surface membrane proteins from yeast endosomes is regulated by ubiquitinated Ist1

“…The physical interactome was acquired from YeastMine (Balakrishnan et al, 2012) Tat2 recycling assay Tryptophan auxotroph (trp1∆) yeast cells were grown to mid log-phase before being spotted out across a 10fold serial dilution and grown on plates of indicated Tryptophan concentrations. Yeast growth was normalised from a wild-type control on the same plate and used the calculate the growth difference, as previously described (Paine et al, 2021).…”