Membrane traffic in the endomembrane system is well characterized at the level of components, but crucial aspects of
the engineering logic of this system remain obscure. Definitions of endomembrane system compartments are often fuzzy,
and knowledge of the directionalities and functions of membrane traffic pathways is incomplete. A particularly enigmatic
organelle is the Golgi apparatus. Studies of yeast cells indicate that Golgi cisternae are transient, maturing structures, with
resident Golgi proteins distributing in a polarized manner across cisternae of different ages. The Golgi recycles components
internally and also communicates extensively with other endomembrane system organelles, but the links between
membrane traffic and Golgi organization are poorly understood. We propose that the Golgi can be productively viewed
as a set of maturing cisternae, with various membrane traffic pathways being switched on and off in an orderly way
during cisternal maturation. Our goal is to elucidate these Golgi-associated membrane traffic pathways and to dissect
the molecular logic circuit that controls them.
We use budding yeasts as an experimental system. The secretory pathway in Saccharomyces cerevisiae has an
unusual organization: non-stacked Golgi cisternae are scattered throughout the cytoplasm, and based on our recent work,
the trans-Golgi network (TGN) serves as an early endosome. These properties simplify the analysis of individual maturing
cisternae by 4D fluorescence microscopy. By determining the kinetic signatures of proteins as they arrive and depart
during cisternal maturation in wild-type or mutant cells, we can obtain novel insights. Recent discoveries include: (1) COPI
vesicles mediate recycling of early but not late Golgi proteins. (2) The AP-1 clathrin adaptor is restricted in yeast to the
TGN. This result, taken together with prior work from other groups, implies that AP-1 mediates intra-Golgi recycling
downstream of COPI. (3) As revealed by our development of a regulatable fluorescent secretory cargo that can be
visualized in maturing cisternae, AP-1 has an unexpected ability to promote intra-Golgi recycling of this secretory cargo.
(4) In unpublished work, AP-1 cooperates with the clathrin adaptor Ent5 to drive two sequential pathways of intra-Golgi
recycling. Transmembrane proteins that recycle by the various COPI- or AP-1-dependent pathways become concentrated
in different cisternae, thereby creating the polarized distribution of proteins across the Golgi.
Our ongoing efforts with S. cerevisiae are aimed at a molecular characterization of these membrane traffic
pathways. We plan to assign roles in specific pathways to individual vesicle tethers, SNAREs, and lipid metabolism
processes. In addition, we will identify functional connections that coordinate the timing of the different pathways.
A newer project employs cultured mammalian cells. We will use imaging and genome editing to revisit three
phenomena that are seemingly at odds with the cisternal maturation concept: nonlinear cargo exit from the Golgi,
exchange of secretory cargoes between Golgi ribbons, and retention of aberrant proteins in the TGN. Those phenomena
can potentially all be explained by a conserved pathway involving AP-1-dependent recycling of secretory cargoes. Our
ambition is to achieve a unified understanding of how the secretory pathway operates in both yeast and mammalian cells.