Extended Data Fig. 3: Mutagenesis of the non-catalytic domains of Vms1.

a, SDS lysates of cells were immunoblotted with anti-haemagglutinin antibodies. Ponceau S staining of the blot shows the equivalency of the extracts. The RR mutant is R313A, R314A. The DNKR mutant contains the following four mutations in the zinc-finger domain: D94A, N99A, K101A, and R102A. The ∆VIM mutant is deleted for amino acids 622–625. b, Serial tenfold dilutions of wild-type and vms1Δ cells transformed with the indicated vms1 alleles were spotted on YPD plates containing 10 or 25 ng ml⁻¹ cycloheximide and incubated at 30 °C for 3 days. Data are representative of three biological replicates. c, Native lysates (10 A₂₆₀ units each) were subjected to sucrose gradient analysis. In each case fractions 10–23 were resolved and immunoblotted to detect protein A and Rpl3. Identical exposures are shown for all panels. d, Native lysates of vms1Δ cells expressing wild-type Vms1–HA3 or Vms1–HA3-DNKR were subjected to sucrose gradient analysis. Fractions were immunoblotted for Vms1 and Rpl3. MW, molecular weight marker. All western blot data are representative of two biological replicates. Gel source images are available in Supplementary Fig. 1.