Morphology and Dynamics of Clathrin/GGA1-coated Carriers Budding from theTrans-Golgi Network

Abstract

Sorting of transmembrane proteins and their ligands at various compartments of the endocytic and secretory pathways is mediated by selective incorporation into clathrin-coated intermediates. Previous morphological and biochemical studies have shown that these clathrin-coated intermediates consist of spherical vesicles with a diameter of 60–100 nm. Herein, we report the use of fluorescent imaging of live cells to demonstrate the existence of a different type of transport intermediate containing associated clathrin coats. Clathrin and the adaptors GGA1 and adaptor protein-1, labeled with different spectral variants of the green fluorescent protein, are shown to colocalize to the trans-Golgi network and to a population of vesicles and tubules budding from it. These intermediates are highly pleiomorphic and move toward the peripheral cytoplasm for distances of up to 10 μm with average speeds of ∼1 μm/s. The labeled clathrin and GGA1 cycle on and off membranes with half-times of 10–20 s, independently of vesicle budding. Our observations indicate the existence of a novel type oftrans-Golgi network-derived carriers containing associated clathrin, GGA1 and adaptor protein-1 that are larger than conventional clathrin-coated vesicles, and that undergo long-range translocation in the cytoplasm before losing their coats.