Journal: Molecular Biology of the Cell
Article Title: A Mutant of Arp2p Causes Partial Disassembly of the Arp2/3 Complex and Loss of Cortical Actin Function in Fission Yeast
Figure Lengend Snippet: Arp2p and Arp3p are ATP-binding proteins. (A) Arp2p and Arp3p were labeled by 8-azido-[α- 32 P]ATP. Wild-type cells carrying pRMH42 arp2 + (KGY2224), pRMH42 arp2 -E316K (KGY2225), or pRMH42 arp2 -T12A (KGY2226) were grown to midlog phase in minimal medium containing thiamine at 25°C, washed to remove the thiamine, and then incubated for 22 h at 25°C to induce expression of the myc-tagged Arp2 proteins. After cell lysis under native conditions, the myc-tagged Arp2 proteins were immunoprecipitated and then mixed with 2 μM 8-azido-[α- 32 P]ATP. Samples were subjected to UV irradiation to promote cross-linking between Arp2p and 8-azido-[α- 32 P]ATP. After irradiation, a fraction of each sample was denatured by boiling, and a second immunoprecipitation was performed with the use of either anti-Arp2 or anti-Arp3 antibodies to distinguish between myc-Arp2p and Arp3p. Cold ATP was added to a final concentration of 500 μM in competition experiments. Labeled proteins were resolved by SDS-PAGE and autoradiography. (B) Immunoprecipitation of myc-tagged Arp2 proteins from lysates used in ATP-binding assays with the 9E10 anti-myc mAb. (Lane 1) Wild type (KGY28); (lane 2) pRMH42 arp2 + (KGY2224); (lane 3) pRMH42 arp2 -E316K (KGY2225); (lane 4) pRMH42 arp2 -T12A (KGY2226). Immunoblots were probed with 9E10 anti-myc antibody to detect tagged Arp2. (C) After induction of myc-tagged wild-type Arp2 (KGY2224) and Arp2-E316K (KGY2225) in wild-type cells for 20 h at 32°C, thiamine was added back to the culture medium to repress expression of the tagged Arp2 proteins from the nmt1 promoter. Lysates were prepared from cells collected hourly after transcriptional repression, and the myc-tagged Arp2 proteins, along with endogenous Arp2p (loading control), were detected by immunoblotting with the use of anti-Arp2 antibodies. (D) myc-tagged Arp2 proteins were immunoprecipitated from native lysates and resolved by SDS-PAGE. (Lane 1) Wild-type (KGY28); (lane 2) pRMH42 arp2 + (KGY2224); (lane 3) pRMH42 arp2 -E316K (KGY2225); (lane 4) pRMH42 arp2 -T12A (KGY2226). The presence of coimmunoprecipitated Arp3p was detected by immunoblotting with the use of anti-Arp3p antibodies.
Article Snippet: Photoaffinity labeling was carried out by mixing immunoprecipitated protein and 2 μM 8-azido-[α-32 P]ATP (20 Ci/mmol; ICN Pharmaceuticals) in a total volume of 100 μl.
Techniques: Binding Assay, Labeling, Incubation, Expressing, Lysis, Immunoprecipitation, Irradiation, Concentration Assay, SDS Page, Autoradiography, Western Blot