A, Comparison of the sequence of Cin4p with that of four human ARF-like proteins, Arl2-5. B, Interaction of Arl2-5 with cofactor D. Analysis on 12% SDS gels of immune precipitates formed by reaction of ³⁵S-labeled Arls with cofactor D and immunoprecipitated with anticofactor D antibody. T, Control translation reactions; PI, immune precipitates formed by preimmune antisera; I, immune precipitates formed by immune antisera. The faster migrating Arl2 band contained in the doublet generated by in vitro translation probably represents internal initiation of Arl2 at Met¹⁰.

A, Analysis by gel filtration of the products of transcription/translation reactions programed with Arl2 and incubated with BSA (as a control; closed circles) or with cofactor D (open triangles). The position of molecular mass markers (left to right: thyroglobulin, 670 kD; bovine IgG, 158 kD; chicken ovalbumin, 44 kD; equine myoglobin, 17 kD) is shown (closed triangles). B and C, Analysis by 12% SDS-PAGE of the fractions shown in B. The peak comigrating with the ovalbumin marker is hemoglobin, which is an endogenous product of the reticulocyte transcription/translation cocktail. Molecular mass markers are shown at the left. D, Autoradiogram of a 12% SDS polyacrylamide gel of the products of an immune precipitation reaction done with anticofactor D antibody and material contained in the 160–200-kD peak generated in a reaction containing added cofactor D. PI, Preimmune antisera; and I, immune antisera. E, Differential binding of translated cofactor D to Arl2 mutant proteins. His-tagged Arl2 proteins were incubated with radiolabeled translated cofactor D and complexes were isolated on an affinity resin. Bound material was analyzed by SDS-PAGE, followed by autoradiography. F, An Arl2 variant containing a mutation in the putative effector loop fails to bind cofactor D. HA-tagged wild-type Arl2 and the corresponding mutations T47A and F50A were translated in vitro, incubated with cofactor D, and immunoprecipitated with an anticofactor D antibody, and the immunoprecipitated material resolved by SDS-PAGE. T, In vitro translation product; PI, preimmune antisera; and I, immune antisera.

A, Arl2, but not Arl3, suppresses the cofactor-induced GTPase activity of tubulin. Relative rates of GTP hydrolysis in reactions containing tubulin, cofactors C, D, and E, and the molar excess (with respect to cofactor D) of Arl 2 or Arl3 shown. Each relative rate was calculated as an average from two or more independent experiments. B, Arl2 prevents the interaction of cofactor D with native tubulin. Purified native tubulin dimers ³⁵S-labeled in the β-subunit by in vitro translation were incubated with cofactor D in the absence or presence of either GST (as a control) or a 5- or 15-fold molar excess (with respect to cofactor D) of purified Arl2. Reaction products were analyzed on a 4% nondenaturing polyacrylamide gel and visualized by autoradiography. Arrows (top to bottom) show the position of Arl2/β-tubulin/cofactor D complex, β-tubulin/cofactor D complex, and native tubulin dimers, respectively.

A, Arl2, but not Arl3, suppresses the cofactor-induced GTPase activity of tubulin. Relative rates of GTP hydrolysis in reactions containing tubulin, cofactors C, D, and E, and the molar excess (with respect to cofactor D) of Arl 2 or Arl3 shown. Each relative rate was calculated as an average from two or more independent experiments. B, Arl2 prevents the interaction of cofactor D with native tubulin. Purified native tubulin dimers ³⁵S-labeled in the β-subunit by in vitro translation were incubated with cofactor D in the absence or presence of either GST (as a control) or a 5- or 15-fold molar excess (with respect to cofactor D) of purified Arl2. Reaction products were analyzed on a 4% nondenaturing polyacrylamide gel and visualized by autoradiography. Arrows (top to bottom) show the position of Arl2/β-tubulin/cofactor D complex, β-tubulin/cofactor D complex, and native tubulin dimers, respectively.

Overexpression of cofactors D or E causes microtubule destruction. Double-label immunofluorescence of HeLa cells transfected with pGFP alone (as a control: A–D), pGFP-D (E–H), or pGFP-E (I–L). Microtubules are shown in red, detected with either an anti–α-tubulin antibody (B, F, and J) or an anti–β-tubulin antibody (D, H, and L).

Overexpression of cofactors D or E results in the loss of tubulin dimers. Double-label immunofluorescence of HeLa cells transfected with pGFP-D (A–D) or pGFP-E (E–H) and treated with nocodazole before fixation. α- or β-tubulin was detected with an anti–α- (B and F) or anti–β-tubulin (D and H) mAb. Note the virtual disappearance of detectable α-tubulin signal in cells transfected with pGFP-D or pGFP-E (B and F), whereas there is retention of a strong β-tubulin signal in cells transfected with pGFP-D (D), and a weaker β-tubulin signal in cells transfected with pGFP-E (H).

Cofactor D/β-tubulin complexes can be immunoprecipitated from cells transfected with pGFP-D, but no corresponding stable α-tubulin containing complexes can be isolated from cells transfected with pGFP-E. Extracts prepared from cells transfected with pGFP-D or pGFP-E were incubated with an anti-GFP antibody and the immune precipitates analyzed by Western blotting with an anti-GFP antibody (top), an anti–α-tubulin antibody (middle) or an anti–β-tubulin antibody (bottom). An extract from untransfected cells was used on the Western blot as a control. Note the detection of β-tubulin from the pGFP-D transfected cell extract, in contrast to the lack of detectable α-tubulin from the pGFP-E transfected cell extract.

Expression of Arl2 rescues microtubules from destruction by overexpression of cofactor D. Triple label immunofluorescence of HeLa cells transfected with pGFP-D and either pHA-Arl2 (A–C) or pHA-Cdc42 (D–F). HA-Cdc42 and HA-Arl2 (Pai et al. 1989), detected with an anti-HA antibody, are shown in blue; microtubules (detected with an anti–α-tubulin antibody) are shown in red. Note that (in contrast to transfected cells expressing GFP-D alone, see Fig. 3) transfected cells coexpressing GFP-D and Arl2 have a normal microtubule phenotype.

Arl2 forms a complex with cofactor D in vivo. A, Western blot analyses of extracts prepared from cells cotransfected with pGFP-D and pHA-Arl2. Extracts were incubated without (−) or with (+) the cross-linking reagent BS3 before resolution by SDS-PAGE. Detection was with either anti-HA antibody (left), or anti-GFP antibody (right). Arrows highlight the band corresponding to the cross-linked Arl2-cofactor D product. B, The cross-linked product in cells cotransfected with p-GFP-D and HA-Arl2 contains both Arl2 and cofactor D. An extract from cells cotransfected with p-GFP-D and pHA-Arl2 was subjected to cross-linking with BS3 and incubated with a preimmune (PI) or immune (I) anticofactor D antibody. Immunoprecipitated material was analyzed by Western blotting using an anti-HA antibody. Molecular weight markers are shown on the left.

Model depicting the action of Arl2 in the reactions involved in the assembly of the tubulin heterodimer and modulation of its guanine nucleotide state. The chaperonin CCT is shown in orange, prefoldin/GimC is in yellow, and cofactors are denoted by red letters.