Figure 4.
Atg9 is associated with membranous structures. (A) Most of the Atg9 is in a low speed supernatant fraction. Spheroplasts from Atg9-PA pep4Δ (FRY172) cells were lysed osmotically and fractionated at 13,000 xg for 12 min into total (TOT), supernatant (S13), and pellet (P13) fractions. After trichloroacetic acid precipitation, proteins were separated by SDS-PAGE and revealed by Western-blot using antibodies and antisera against protein A (PA), Pgk1, porin, Pho8 and Vma4. (B) Atg9 is always associated with membranes. S13 and P13 fractions prepared as in (A) were subjected to the following treatments: lysis buffer (-), 1 M KCl (KCl) and 1% Triton X-100 (TX-100) before being centrifuged at 100,000 xg for 30 min. Proteins from the supernatant (S100) and pellet (P100) fractions were analyzed as in (A).