Figure 4Time-lapse images of agarose-embedded cells were taken at RT with supply of fresh PM Glc+ura medium throughout one budding cycle. Only (F) shows fixed cells immunostained for Sed5p. All cells produced the β'-COPVN•Dsl3pVC BiFC pair. Most strains contained plasmids encoding different organelle markers as indicated below. BiFC signals are pseudocolored green, organelle markers are pseudocolored red. Scale bar 5 µm.A. RFPHDEL, ER marker Time-lapse images of agarose-embedded cells were taken at RT with supply of fresh PM Glc+ura medium throughout one budding cycle. Only (F) shows fixed cells immunostained for Sed5p. All cells produced the β'-COPVN•Dsl3pVC BiFC pair. Most strains contained plasmids encoding different organelle markers as indicated below. BiFC signals are pseudocolored green, organelle markers are pseudocolored red. Scale bar 5 µm.B. Sec13pRFP, ER exit sites marker Time-lapse images of agarose-embedded cells were taken at RT with supply of fresh PM Glc+ura medium throughout one budding cycle. Only (F) shows fixed cells immunostained for Sed5p. All cells produced the β'-COPVN•Dsl3pVC BiFC pair. Most strains contained plasmids encoding different organelle markers as indicated below. BiFC signals are pseudocolored green, organelle markers are pseudocolored red. Scale bar 5 µm.C. RFPSed5p, cis-Golgi markers (genomic) Time-lapse images of agarose-embedded cells were taken at RT with supply of fresh PM Glc+ura medium throughout one budding cycle. Only (F) shows fixed cells immunostained for Sed5p. All cells produced the β'-COPVN•Dsl3pVC BiFC pair. Most strains contained plasmids encoding different organelle markers as indicated below. BiFC signals are pseudocolored green, organelle markers are pseudocolored red. Scale bar 5 µm.D. CFPGos1p, medial Golgi marker Time-lapse images of agarose-embedded cells were taken at RT with supply of fresh PM Glc+ura medium throughout one budding cycle. Only (F) shows fixed cells immunostained for Sed5p. All cells produced the β'-COPVN•Dsl3pVC BiFC pair. Most strains contained plasmids encoding different organelle markers as indicated below. BiFC signals are pseudocolored green, organelle markers are pseudocolored red. Scale bar 5 µm.E. Sec7pCFP, trans-Golgi marker Time-lapse images of agarose-embedded cells were taken at RT with supply of fresh PM Glc+ura medium throughout one budding cycle. Only (F) shows fixed cells immunostained for Sed5p. All cells produced the β'-COPVN•Dsl3pVC BiFC pair. Most strains contained plasmids encoding different organelle markers as indicated below. BiFC signals are pseudocolored green, organelle markers are pseudocolored red. Scale bar 5 µm.F. Individual fixed cells carrying the β'-COPVN•Dsl3pVC BiFC pair along with trans-Golgi marker CFPSec7p were used for this panel. The β'-COPVN•Dsl3pVC BiFC signals are pseudocolored white. Immunostaining was used to visualize the cis-Golgi marker Sed5p (red). The trans-Golgi marker CFPSec7p is pseudocolored green. Time-lapse images of agarose-embedded cells were taken at RT with supply of fresh PM Glc+ura medium throughout one budding cycle. Only (F) shows fixed cells immunostained for Sed5p. All cells produced the β'-COPVN•Dsl3pVC BiFC pair. Most strains contained plasmids encoding different organelle markers as indicated below. BiFC signals are pseudocolored green, organelle markers are pseudocolored red. Scale bar 5 µm.G. CFPRer1, COPI cargo Time-lapse images of agarose-embedded cells were taken at RT with supply of fresh PM Glc+ura medium throughout one budding cycle. Only (F) shows fixed cells immunostained for Sed5p. All cells produced the β'-COPVN•Dsl3pVC BiFC pair. Most strains contained plasmids encoding different organelle markers as indicated below. BiFC signals are pseudocolored green, organelle markers are pseudocolored red. Scale bar 5 µm.H. CFPSec4p, marker for secretory vesicles