IEM analysis of wild-type cells expressing Atg9-GFP. Atg9-GFP (MMY067) cells were grown to log phase and processed for IEM as described in Materials and methods. Preparations shown in A–D were immunogold labeled only for GFP, whereas those presented in E were double immunogold labeled for GFP and Ape1. (A) Overview of a labeled cell section. (B) Insets of A illustrating an Atg9-positive cluster of vesicles and tubules. (C) Atg9-containing membranous arrangements (arrow) adjacent to mitochondria (≤50 nm distance). (D) Micrograph showing a GFP-labeled cell with two Atg9 clusters (marked by an arrow and an arrowhead), one of which (arrowhead) associated with an electron-dense structure with a 100–150 nm diameter (broken lines). (E) The circular electron-dense structures found in close proximity to the Atg9 clusters and labeled for Ape1 are Cvt complexes (broken lines). The dashed cisterna emphasizes the Cvt complex surface not associated with Atg9-containing membranes. D and E are also shown in Fig. S1 (A and B) without broken lines for clarity, while additional examples of Atg9 clusters are presented in Fig. S1, C–H. The size of the gold particles is indicated on the top of each picture. CW, cell wall; M, mitochondria; MVB, multivesicular body; N, nucleus; PM, plasma membrane; V, vacuole. Bars, 200 nm.