biostudies-literatureUnknownKrahn AHNCI NIH HHSNational Institutes of HealthNIGMS NIH HHSThe traffic pathways that recycle resident Golgi proteins during cisternal maturation are not completely defined. We addressed this challenge using the yeast <i>Saccharomyces cerevisiae</i>, in which maturation of individual cisternae can be visualized directly. A new assay captures a specific population of Golgi-derived vesicles at the bud neck, thereby revealing which resident Golgi proteins are carried as cargo in those vesicles. This method supplies evidence for at least three classes of intra-Golgi vesicles with different cargo compositions. Consistent with our previously published data, one class of vesicles mediates a late pathway of intra-Golgi recycling with the aid of the AP-1 and Ent5 clathrin adaptors, and a second class of vesicles mediates an early pathway of intra-Golgi recycling with the aid of the COPI vesicle coat. Here, we identify another COPI-dependent pathway of intra-Golgi recycling and show that it operates kinetically between the two previously known pathways. Thus, intra-Golgi recycling is mediated by multiple COPI-dependent pathways followed by a clathrin-dependent pathway.bioRxiv : the preprint server for biology2025.09.20.677526https://www.ebi.ac.uk/biostudies/studies/S-EPMC12458367biostudies-literatureCOPI-dependent intra-Golgi recycling at an intermediate stage of cisternal maturation.PMC12458367T32 GM007183R35 GM144050P30 CA014599Glick BSLee-Smith CFPantazopoulou AAustin JJohnson NKrahn AHfalseCOPI-dependent intra-Golgi recycling at an intermediate stage of cisternal maturation.The traffic pathways that recycle resident Golgi proteins during cisternal maturation are not completely defined. We addressed this challenge using the yeast <i>Saccharomyces cerevisiae</i>, in which maturation of individual cisternae can be visualized directly. A new assay captures a specific population of Golgi-derived vesicles at the bud neck, thereby revealing which resident Golgi proteins are carried as cargo in those vesicles. This method supplies evidence for at least three classes of intra-Golgi vesicles with different cargo compositions. Consistent with our previously published data, one class of vesicles mediates a late pathway of intra-Golgi recycling with the aid of the AP-1 and Ent5 clathrin adaptors, and a second class of vesicles mediates an early pathway of intra-Golgi recycling with the aid of the COPI vesicle coat. Here, we identify another COPI-dependent pathway of intra-Golgi recycling and show that it operates kinetically between the two previously known pathways. Thus, intra-Golgi recycling is mediated by multiple COPI-dependent pathways followed by a clathrin-dependent pathway.2025-01-01T00:00:00Z2025 Sep2026-05-04T03:21:01.422Z2026-05-04T03:14:25.638ZS-EPMC124583674100080410.1101/2025.09.20.677526