100-kD Proteins of Golgi- and Trans-Golgi Network-Associated Coated Vesicles Have Related but Distinct Membrane Binding Properties

• Published in: The Journal of cell biology 1992-06, Vol.117 (6), p.1171-1179
• Main Authors: Wong, Darren H., Brodsky, Frances M.
• Format: Article
• Language: English
• Subjects: Adaptor Protein Complex alpha Subunits; Adaptor Protein Complex gamma Subunits; Adaptor Proteins, Vesicular Transport; Animals; Biological and medical sciences; Brefeldin A; Capsid proteins; Cell Line; Cell lines; Cell membranes; Cell structures and functions; Cells; Cellular biology; Clathrin coated vesicles; Coated Pits, Cell-Membrane - metabolism; Coated vesicles; Coatomer Protein; Cyclopentanes - pharmacology; Cytoplasm - metabolism; Cytosol; Fluorescent Antibody Technique; Fundamental and applied biological sciences. Psychology; Golgi apparatus; Golgi Apparatus - metabolism; Golgi Apparatus - ultrastructure; guanine nucleotide-binding protein; Hepatocytes; interaction; Intracellular Membranes - metabolism; MDBK cells; Microtubule-Associated Proteins - metabolism; Molecular and cellular biology; P branes; Protein Binding; Proteins; Proteins - metabolism
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.117.6.1171

The 100-110-kD proteins (α, β-, β′-, and γ-adaptins) of clathrin-coated vesicles and the 110-kD protein (β-COP) of the nonclathrin-coated vesicles that mediate constitutive transport through the Golgi have homologous protein sequences. To determine whether homologous processes are involved in assembly of the two types of coated vesicles, the membrane binding properties of their coat proteins were compared. After treatment of MDBK cells with the fungal metabolite Brefeldin A (BFA), β-COP was redistributed to the cytoplasm within 15 s, γ-adaptin and clathrin in the trans-Golgi network (TGN) dispersed within 30 s, but the α-adaptin and clathrin present on coated pits and vesicles derived from the plasma membrane remained membrane associated even after a 15-min exposure to BFA. In PtK1 cells and MDCK cells, BFA did not affect β-COP binding or Golgi morphology but still induced redistribution of γ-adaptin and clathrin from TGN membranes to the cytoplasm. Thus BFA affects the binding of coat proteins to membranes in the Golgi region (Golgi apparatus and TGN) but not plasma membranes. However, the Golgi binding interactions of β-COP and γ-adaptin are distinct and differentially sensitive to BFA. BFA treatment did not release γ-adaptin or clathrin from purified clathrin-coated vesicles, suggesting that their distribution to the cytoplasm after BFA treatment of cells was due to interference with their rebinding to TGN membranes after a normal cycle of disassembly. This was confirmed using an in vitro assay in which γ-adaptin binding to TGN membranes was blocked by BFA and enhanced by GTPγS, similar to the binding of β-COP to Golgi membranes. These results suggest the involvement of GTP-dependent proteins in the association of the 100-kD coat proteins with membranes in the Golgi region of the cell.