Mup1-GFP recycling occurs from a Vps4-Ist1 endosome. (A and B) 4D Airyscan microscopy of WT cells co-expressing Mup1-GFP and Sec7-mCherry (upper) and Vps4-mCherry (lower) following a 30-s 20 µg/ml methionine pulse and subsequent SC-Met chase period over short 2–4 s (A) and long 30–60 s (B) imaging intervals. (C) Quantification of Pearson’s correlation coefficients between intercellular Mup1-GFP and either Sec7-mCherry (gray) or Vps4-mCherry (green) signal from steady state images acquired at indicated times during methionine pulse-chase (n = >27 cells), *, P < 0.0001 from unpaired Student’s t test, individual values are included as jitter plot over histograms and represent n = 61–79 cells per condition. (D) Mander’s overlap coefficient between Mup1-GFP and either Sec7-mCherry (gray) or Vps4-mCherry (green) from representative real-time methionine pulse-chase imaging experiment. (E and F) Time-lapse Airyscan micrographs of Mup1-GFP and Sec7-mCherry (E) or Vps4-mCherry (F) were analyzed by Zen Black colocalization software, and regions of Manders overlap (not signal colocalization) above background that were detected are depicted in white, with zoomed-in representations of the PM and endosome. Scale bar, 5 μm (white); 0.5 μm (yellow).