Purification and architecture of the ubiquitous Wave complex

Abstract

The Wave proteins are major activators of the Arp2/3 complex. The ubiquitous Wave-2 is required for actin polymerization at the leading edge of migrating cells. Here we purify Wave-2 from HeLa cells. Five proteins, Sra, Nap, Wave-2, Abi, and Hspc, are copurified, indicating that they form a tight complex. These proteins are only present in the complexed form, with the exception of Hspc, which displays a free pool. We reconstitute the Wave-2 complex by cotranslating in vitro the five subunits and use this system together with specific immunoprecipitations to study the molecular architecture of the complex. The complex is organized around a core of Nap and Abi. Sra is a peripheral subunit recruited on the Nap side, whereas the Wave and Hspc subunits are recruited on the Abi side of the core.

Fig. 1.

Purification of the Wave-2-containing complex from HeLa cells. After four steps of chromatography (depicted on the left), the final fractions obtained after the sucrose gradient were loaded on 5–15% gradient polyacrylamide gel. After SDS/PAGE, the gel was silver-stained, and the bands were excised and analyzed by mass spectrometry. In addition to the four clear bands, Hspc was not detected by the staining of the gel but was clearly detected when the complete fraction was analyzed by mass spectrometry.

Fig. 2.

Distribution of the subunits and reconstitution of the Wave-2 complex. (A) A HeLa extract was loaded on a sucrose gradient, and the fractions were immunoblotted with antibodies targeting the different subunits. The complex peaks at ≈11 Swedbergs (fractions 13 and 14) where all five proteins could be detected. (B) Reconstitution of the complex by in vitro translation. The genes encoding the five subunits were cotranslated in vitro in the presence of [³⁵S]methionine, and the mixture was separated on a sucrose gradient. Nap is the limiting component in the mixture and is highly enriched at the size of the native complex, 11 S. The other subunits also were incorporated in the reconstituted complex at 11 S.

Fig. 3.

Interactions between subunits. All possible pairs of subunits were cotranslated in vitro in the presence of [³⁵S]methionine, one subunit being untagged (+) and the other being tagged at the N terminus by HA epitopes (⊕). The input and HA immunoprecipitates were resolved by SDS/PAGE and autoradiographed. The immunoprecipitated subunits are indicated on the autoradiograph by arrows. The lanes demonstrating a significant interaction (over the negative control) are numbered, and the interactions are depicted on a diagram on the right.

Fig. 4.

Reconstitution of all possible complexes lacking one subunit. The five subunits were cotranslated in vitro in the presence of [³⁵S]methionine. The HA-tagged subunit is indicated by ⊕, and the omitted subunit is indicated by –. The presence of all of the untagged subunits is not indicated for the sake of clarity. For each reaction, two lanes are shown, the input on the left and the HA immunoprecipitate on the right.