Secretory pathway kinetics and in vivo analysis of protein traffic from the Golgi complex to the cell surface

Eukaryotic cells, from yeast to human, have the ability to synthesize and secrete highly processed and complex molecules. This enables them to exquisitely modify their cell surface and extracellular space, and thereby to respond to stimuli in their environment and to communicate with other cells. This property depends on the correct functioning of the secretory pathway, which is comprised of distinct membranebound compartments (including the endoplasmic reticulum [ER], Golgi complex and plasma membrane) and vesicle/tubule transport intermediates. Newly synthesized protein and lipid enter this pathway in the ER and are transported to the Golgi complex for further processing and maturation by glycolipid and glycoprotein trimming enzymes. On reaching the trans-Golgi network, they are sorted and packaged into post-Golgi transport intermediates that deliver them to the plasma membrane. Cell biologists have made significant progress in elucidating the biochemical requirements for secretory transport and the role of transport intermediates (1, 2). Less understood are the morphological and kinetic properties of secretory traffic, including how transport intermediates are formed and their path and fate during transport. In addition, it is not clear how long cargo resides in a particular compartment and the rate of cargo influx and efflux out of a given compartment. This lack of understanding is largely because of the inherent limitations of traditional approaches. For example, biochemical assays using either in vitro or permeabilized systems (3, 4) often have difficulty identifying and/or characterizing intermediate steps because the amount of cargo carried by transport intermediates within the secretory pathway is usually quite small. Immunofluorescence and electron microscopy approaches, which can be used to identify carriers, provide only static snapshots of cells and therefore are unable to reveal the origin/fate of transport intermediates and their dynamic properties. A recent technique that overcomes many of the limitations inherent in studies of in vitro or fixed specimens exploits the intrinsic fluorescence of the green fluorescent protein (GFP) as a tag for the production of fusion proteins (5). GFP fusion proteins are easily expressed and visualized within living cells. They are remarkably photostable to repetitive imaging using low illumination levels and, in many cases, do not perturb normal trafficking or function of the parent protein (6, 7). They also can be quantitated by correlating their fluorescence to a known standard solution of GFP molecules (8, 9). It is not surprising, therefore, that GFP fusion proteins have been used as fluorescent reporters in a wide variety of applications in living cells, including timelapse imaging studies to follow changes in protein distribution (10, 11), quantitative imaging experiments to measure the amount of protein localized at a particular site (12, 13), and biophysical assays such as photobleaching to measure protein mobility (14). To study protein trafficking through the secretory pathway, we have used a fusion protein where GFP was attached to the temperature sensitive protein known as ts045 VSVG from vesicular stomatitis virus (11). The ts045 VSVG protein is a type I transmembrane protein that has been widely used for studying secretory membrane traffic because it contains a mutation that leads to its reversible misfolding and retention in the ER at 40°C (15–19). On temperature reduction (e.g., to 32°C) the protein folds correctly and is exported out of the ER and through the secretory pathway as a synchronous pool. This property makes ts045 VSVG an ideal tool for following protein traffic within cells. Addition of the GFP tag to the cytoplasmic tail of ts045 VSVG did not alter either the folding or transport properties of this protein (11, 19). Recently, GFP-tagged ts045 VSVG (VSVG-GFP) was used to visualize ER-to-Golgi traffic in living cells (11, 19). Release from the 40°C temperature block by shifting to 32°C resulted in the ER-localized chimera first concentrating in large, pleiomorphic pre-Golgi structures (known as the