FIGURE 4:

Tubulin chaperones help recycle tubulin at low temperatures. (A) Model of the tubulin biogenesis pathway (adapted from Nithianantham et al., 2015; Tian et al., 1997). (B, C) Tenfold dilution series of null strains for the prefoldin (B), CCT (B), and TBCs (C) indicated at left were spotted to rich medium or rich medium supplemented with 5 μg/ml benomyl and grown at the indicated temperature for the indicated time. (D) Representative field of alf1Δ cells expressing GFP-Tub1 incubated at 30°C, 25 min after shifting to 4°C, and 20 and 40 s after shifting back to 30°C from 4°C. (E) Proportion of wild-type (solid line) and alf1Δ (dotted line) with astral microtubules present after shifting from 4°C to 30°C for the indicated time. Each data point represents a proportion calculated from data pooled from three separate experiments with at least 266 cells analyzed for each timepoint. Error bars are standard error of proportion. (F) Tenfold dilution series of strain indicated at left were spotted to rich medium or rich medium supplemented with 5 μg/ml benomyl and grown at the indicated temperature for the indicated time. (G) Tenfold dilution series of strain carrying the plasmid indicated at left were spotted to rich medium or rich medium supplemented with 5 μg/ml benomyl and grown at the indicated temperature for the indicated time. (H) Western blot of protein lysate from wild-type or tub1-R265C cells carrying the indicated plasmid incubated at 30°C or 15°C for 24 h and probed for α-tubulin and Zwf1.