Figure 1.

Examination of the interactions between Vps5p and the other retromer components. Approximately 6 OD_{600 nm} equivalents of spheroplasted cells were labeled with [³⁵S]methionine for 20 min and then chased for 40 min. The cells were lysed in cytosol buffer containing 0.5% Triton X-100. The lysate was cleared by centrifugation for 5 min at 13,000 × g and transferred to a fresh tube. Vps5p was immunoprecipitated under native conditions from the lysate by using an affinity purified polyclonal antisera (Horazdovsky et al., 1997). After several washes the samples were boiled in urea cracking buffer and a fresh lysate was generated. Vps35p, Vps17p, and Vps29p were then simultaneously immunoprecipitated after which the lysate was reimmunoprecipitated with antibodies against both Vps5p and Vps26p. This experiment provides a benchmark for further experiments to dissect Vps5p and identify its functional domains.