Defective actin organization in arc35-1 cells. Wt (RH2877) and arc35-1 (RH3429) strains were preincubated for 3 h at either 24 or 37°C, fixed, stained with rhodamine–phalloidin, and visualized either by Nomarski optics or by epifluorescence. Shown are the Wt strain at 24°C (A, B) and 37°C (C, D) and the arc35-1 strain at 24°C (E, F) and 37°C (G, H), respectively. Bar, 5 μm.

Suppression of arc35-1 ts growth by calmodulin. Wt (RH2877) (A), arc35-1 (RH3429) (B), and arp2-1 (RH4164) (C) strains were transformed with the following plasmids: (A) pASZ12, (B) pCMD1, (C) pASZ12::cmd1-226 (actin), (D) pASZ12::cmd1-228 (calmodulin mislocalized), (E) pASZ12::cmd1-239 (nuclear defect), (F) pASZ12::cmd1-231 (bud emergence), (G) pASZ12::cmd1-247 (endocytosis), (H) pASZ12::cmd1-3 (calcium binding). The strains were grown on glucose-selective medium for 3 d at 24°C and for 2 d at 37°C.

Arc35p is required to localize calmodulin to the cortical sites. Wt (RH2877), arc35-1 (RH3429), arp2-1 (RH4164), and arp3-1 (Y90) strains were grown at 24°C and then shifted to 37°C for 10, 30, or 60 min before fixation and staining for calmodulin by indirect immunofluorescence. The staining obtained using preabsorbed α-calmodulin antibody is also shown (PA). Bar, 5 μm.

Coimmunoprecipitation of Arc35p and Cmd1p. (A) To assess the specificity of the Arc35p antibody, Wt cells expressing either endogenous Arc35p or overexpressing Arc35p (RH2877+pASZ12::ARC35) were grown in selective medium and harvested, and proteins were extracted. The extracts were immunoblotted for Arc35p and Cmd1p as a loading control. (B) A Wt strain (RH2877) was grown to logarithmic phase and harvested. Total and native protein extracts were prepared. Protein A-Sepharose (prot. A), protein A-Sepharose bound to preimmune serum (preimm.), or protein A-Sepharose bound to α-Cmd1p serum (α-Cmd1p) was used for immunoprecipitation of native cell extracts in the presence of no calcium or 5 mM calcium. Immunoprecipitates (P) and supernatants (S) were immunoblotted for Arc35p, Cmd1p, or Act1p with α-Arc35p-N, α-Cmd1p, or α-actin antibodies.

Overexpression of calmodulin and distinct calmodulin mutant alleles restores endocytosis in arc35-1. (A) Wt (RH2877) and arc35-1 (RH3429) strains without plasmid or bearing plasmid pCMD1 (+Cmd1p) were assayed for their ability to internalize radioactive α-factor at 24 and 37°C after a preincubation of 20 min at the indicated temperature. (B) Wt (RH2877), arc35-1 (RH3429), and arp2-1 (RH4164) strains without plasmid or overexpressing the indicated mutant calmodulin protein from pASZ12 were assayed for the internalization of radioactive α-factor at 24 or 37°C after a preincubation of 20 min. The results are presented as the percentage of radioactive α-factor internalized per minute during the linear range of the uptake reaction.

Arc35-GFP colocalizes with filamentous actin. (A) A Wt strain expressing Arc35-GFP (RH4161) was visualized with epifluorescence (a) or Nomarski optics (b). Bar, 5 μm. (B) A Wt strain expressing Arc35-GFP (RH4161) was grown at 24°C, stained for F-actin with rhodamine–phalloidin, and visualized either with Nomarski optics (c) or with epifluorescence filters suitable to distinguish between GFP and rhodamine signals. The F-actin staining is shown in a, and the signal of Arc35-GFP is shown in b. Bar, 5 μm.

Sensitivity of arc35-1 expressing various calmodulin mutant proteins to latrunculin A. (A) An arc35-1 strain (RH3429) without plasmid (a, b) or overexpressing Cmd1p from pCMD1 (c, d) was preincubated for 3 h, stained with rhodamine–phalloidin, and visualized with epifluorescence. Bar, 5 μm. (B) Wt (RH2877) and arc35-1 (RH3429) strains bearing the empty control plasmid (pASZ12) or overexpressing the indicated calmodulin mutant protein from pASZ12 were used for determination of the apparent relative sensitivity to latrunculin A.

Two-hybrid interaction between Arc35p and Cmd1p. ARC35 and arc35-1 were cloned in frame to the lexA DNA binding domain of pEG202. CMD1 and the collection of ts cmd1 alleles were cloned in frame behind the B42 transactivation domain of pJG4-5. β-Galactosidase activity indicative of interaction was monitored on X-Gal plates at 37°C supplemented with no calcium or 5 mM calcium.