FIGURE 3:

Isolation and characterization of alp41 ts mutants and the physical interaction between Alp41 and Tbc1. (A) Serial dilution spot test of alp41 ts mutants. Mutants were spotted onto rich media plates containing phloxine B or TBZ and incubated at either 27 or 36°C. Three representative alleles are shown. (B) Microtubule morphology. Wild-type and alp41-5 mutant cells with GFP-labeled Atb2 were incubated at 36°C for 4 and 8 h and observed for microtubule defects. Bottom, representative cells from wild-type and mutant strains. Bar, 5 μm. (C) Quantification of microtubule phenotypes. Three hundred cells were quantified and characterized into five microtubule phenotypes: “spindle defects” showing malformed/dysfunctional mitotic spindle, no microtubules, curled microtubules, short interphase microtubules, or normal microtubules. (D) Immunoprecipitation of Alp41 and Tbc1. IPs were performed using a doubly tagged strain expressing GFP-Alp41 and Tbc1-3HA. First lane, control strain expressing GFP-Alp41 only; second lane, Tbc1-3HA only; third lane, doubly tagged strain. A 30-μg amount of whole-cell extract was loaded. Asterisk, unspecific band detected by antibody. (E) Peptide array assay showing interaction of Tbc1 and Alp41. Recombinant Alp41 and Tbc1 proteins (hexahistidine tagged) were purified from E. coli and incubated with synthesized peptides. Left, immunoblots against an anti-histidine antibody; right, schematic of the interacting regions with the known domains. Numbers on the blot mark the amino acid number at the beginning of the row. Top blot, cellulose membrane spotted with Tbc1 peptide incubated with Alp41 protein. Bottom blot, cellulose membrane spotted with Alp41 peptide incubated with Tbc1 protein. (F) Mutual rescue of tbc1 and alp41 ts mutants. Double mutants of tbc1 and alp41 were spotted onto rich media plates with added phloxine B. Plates were incubated at 27, 34, and 36°C. Two alleles of alp41 (-8 and -14) were crossed with tbc1-11.