Phosphoinositide-mediated clathrin adaptor progression at the trans-Golgi network

Abstract

Clathrin coated vesicles mediate endocytosis and transport between the trans Golgi network (TGN) and endosomes in eukaryotic cells. Clathrin adaptors play central roles in coat assembly, interacting with clathrin, cargo, and membranes. Two major types of clathrin adaptors act in TGN-endosome traffic, Gga proteins and the AP-1 complex. Here we characterize the relationship between Gga proteins, AP-1, and other TGN clathrin adaptors using live cell and superresolution microscopy in yeast. We present evidence that Gga proteins and AP-1 are recruited sequentially in two waves of coat assembly at the TGN. Mutations that decrease phosphatidylinositol 4-phosphate (PI4P) levels at the TGN slow or uncouple AP-1 coat assembly from Gga coat assembly. Conversely, enhanced PI4P synthesis shortens the time between adaptor waves. Gga2p binds directly to the TGN PI4-kinase Pik1p and contributes to Pik1p recruitment. These results identify a PI4P-based mechanism for regulating progressive assembly of adaptor-specific clathrin coats at the TGN.

Conserved role for Gga proteins in phosphatidylinositol 4-kinase localization to the trans -Golgi network

“…3C and Fig. S3B), in agreement with previous results (5). By comparison, a marked delay in GFP-PH OSH1 recruitment was evident in both pik1 mutants, with average peak intensities reached 8.7 s ± 1.0 s (pik1 gga ; P = 7.8 × 10 −5 , n = 51 puncta) and 11.3 s ± 1.9 s (pik1 A-3A ; P = 1.2 × 10 −3 , n = 18 puncta), after the peak of Sec7-mRFP ( Fig.…”

“…In prior work (5), the interaction between Gga2 and Pik1 was mapped to the Gga2-VHS domain, which binds to a region of Pik1 that spans amino acids 80-760. To more precisely define VHS binding sites in this region of Pik1, we assessed direct binding of smaller Pik1 fragments to Gga2-VHS.…”

“…We have described a role for PtdIns4P in controlling sequential assembly of adaptorspecific ccv at the TGN in yeast (5), which correlates with sequential cargo-sorting events (11). The assembly sequence, termed adaptor progression, initiates with a wave of ccv enriched for Gga proteins and Ent3.…”

“…At the TGN, phosphatidylinositol 4-phosphate (PtdIns4P) provides this function. The primary TGN adaptors-the heterotetrameric AP-1 (assembly protein-1) complex, GGA (Golgi-localized, gammaadaptin ear homology, Arf-binding) proteins, and epsin-related proteins (epsinR in mammals, Ent3 and Ent5 in yeast)-preferentially bind PtdIns4P and/or are dependent on PtdIns4P for optimal localization and dynamics (5)(6)(7)(8)(9)(10).…”

Clathrin Terminal Domain-Ligand Interactions Regulate Sorting of Mannose 6-Phosphate Receptors Mediated by AP-1 and GGA Adaptors

“…Acute or sustained loss of AP-1 depletes MPRs from clathrin-coated vesicles and impairs MPR retrieval to the TGN (10,20). Loss of AP-1 from TGN/endosomal membranes in addition results in co-depletion of GGA2 from clathrin-coated structures (21), suggesting that AP-1 and GGA2 are functionally linked (22).…”