The functions of the AP-1 clathrin adaptor have been ambiguous. Our studies of budding yeast revealed that AP-1 mediates intra-Golgi recycling of a subset of late Golgi membrane proteins. Unexpectedly, our data also implicate yeast AP-1 in the intra-Golgi recycling of a soluble fluorescent secretory cargo protein. We will test the hypothesis that AP-1 mediates a conserved intra-Golgi recycling pathway that operates in mammalian cells, and that some secretory cargo proteins can recycle by the AP-1 pathway before exiting the Golgi. Specific Aim #1 is to characterize how yeast AP-1 recycles resident late Golgi proteins from maturing cisternae, and to explore how a soluble secretory glycoprotein is recycled in an AP-1-dependent manner. We will use an AP-1 deletion strain to determine how the yeast secretory pathway can bypass AP-1, and to study the role of this adaptor in intra-Golgi recycling. Sub-Aim #1A is to determine how the yeast secretory pathway can tolerate the absence of AP-1. Our hypothesis is that proteins that normally recycle within the Golgi in AP-1 vesicles can recycle instead in endocytic vesicles that travel from the plasma membrane to the Golgi. We will test this idea by asking whether the combination of an endocytosis block with an AP-1 deletion disrupts Golgi maturation, and by examining whether an AP-1 deletion causes certain late Golgi proteins to remain present in terminally maturing cisternae. Sub-Aim #1B is to determine whether the mannosyltransferase Mnn1 might serve as a ?receptor? to recycle the fluorescent secretory cargo protein. Our hypothesis is that glycans on the secretory cargo protein are kinetically bound to Mnn1, which recycles in AP-1 vesicles. We will test this idea by deleting Mnn1, and by testing whether Mnn1 localization depends on AP-1. Sub-Aim #1C is to identify components that mediate AP-1 vesicle targeting and fusion. Our hypothesis is that the SNAREs Gos1 and Sft1 recycle in AP-1 vesicles and participate in AP-1 vesicle fusion. We will test this idea by characterizing the maturation kinetics of Gos1 and Sft1 in wild-type and AP-1 deletion strains. In addition, we will test putative tethers for their ability to capture AP-1 vesicles. Specific Aim #2 is to test whether AP-1 mediates intra-Golgi and inter-Golgi recycling of secretory cargo proteins in mammalian cells. We will examine the role of AP-1 in phenomena that have been described as challenges to the cisternal maturation model for Golgi function. Sub-Aim #2A is to determine whether the nonlinear kinetics of secretory cargo exit from the mammalian Golgi depend on AP-1. Our hypothesis is that the kinetics of exit from the mammalian Golgi reflect AP-1-dependent recycling. We will test this idea by asking whether inactivation of AP-1 alters Golgi export behavior for soluble and transmembrane secretory cargoes. Sub-Aim #2B is to determine whether Arf1-dependent exchange of secretory cargo proteins between the Golgi ribbons of fused cells depends on AP-1. Our hypothesis is that the relevant role of the Arf1 GTPase is to recruit AP-1, and that the observed inter-Golgi exchange reflects transport in AP-1 vesicles. We will test this idea by asking whether inactivation of AP-1 prevents inter-Golgi transport of a secretory cargo protein.

R01GM137004

2020-04-01

2024-03-31