FIGURE 3.

UBI4 is the predominant source of ubiquitin for zinc-limited cells. A, wild-type (BY4741) and ubi4::KanMX4 cells were grown in zinc-deficient (D; LZM + 1 μm ZnCl₂) or zinc-replete (R; LZM + 100 μm ZnCl₂) medium. The abundance of ubiquitin and a loading control protein (Pgk1) was assayed by immunoblotting (one representative immunoblot is shown). Levels of ubiquitin conjugates (Ub-C) and monomeric ubiquitin (Ub-M) were quantified from three replicates. B, effect of zinc status and ubi4Δ mutation on expression of ubiquitin precursor genes and the ribosomal subunit gene RPL1B. Wild-type (BY4741), ubi4::KanMX4, and ubi4Δ rpt2^E301K (CWM280) cells were grown to log phase in SD medium and then used to inoculate zinc-deficient (ZnD; LZM + 1 μm ZnCl₂) or -replete (ZnR; LZM + 100 μm ZnCl₂) medium at low starting densities. Cultures were maintained in log phase by dilution with fresh media for 24 h. The mRNA abundance of the indicated genes was then determined by RT-qPCR. Target transcript abundance was normalized to the average abundance of three control transcripts (18S rRNA, TAF10, and ACT1). All plotted data points represent the means of three replicates, and error bars denote ±1 S.D. A.U., arbitrary units.