Kinetic Analysis of Secretory Protein Traffic and Characterization of Golgi to Plasma Membrane Transport Intermediates in Living Cells

Abstract

Quantitative time-lapse imaging data of single cells expressing the transmembrane protein, vesicular stomatitis virus ts045 G protein fused to green fluorescent protein (VSVG–GFP), were used for kinetic modeling of protein traffic through the various compartments of the secretory pathway. A series of first order rate laws was sufficient to accurately describe VSVG–GFP transport, and provided compartment residence times and rate constants for transport into and out of the Golgi complex and delivery to the plasma membrane. For ER to Golgi transport the mean rate constant (i.e., the fraction of VSVG–GFP moved per unit of time) was 2.8% per min, for Golgi to plasma membrane transport it was 3.0% per min, and for transport from the plasma membrane to a degradative site it was 0.25% per min. Because these rate constants did not change as the concentration of VSVG–GFP in different compartments went from high (early in the experiment) to low (late in the experiment), secretory transport machinery was never saturated during the experiments.The processes of budding, translocation, and fusion of post-Golgi transport intermediates carrying VSVG– GFP to the plasma membrane were also analyzed using quantitative imaging techniques. Large pleiomorphic tubular structures, rather than small vesicles, were found to be the primary vehicles for Golgi to plasma membrane transport of VSVG–GFP. These structures budded as entire domains from the Golgi complex and underwent dynamic shape changes as they moved along microtubule tracks to the cell periphery. They carried up to 10,000 VSVG–GFP molecules and had a mean life time in COS cells of 3.8 min. In addition, they fused with the plasma membrane without intersecting other membrane transport pathways in the cell. These properties suggest that the post-Golgi intermediates represent a unique transport organelle for conveying large quantities of protein cargo from the Golgi complex directly to the plasma membrane.

Actin filaments are involved in the maintenance of Golgi cisternae morphology and intra‐Golgi pH

“…Abnormal ion gradients and pH coupled to the loss of actin cytoskeleton integrity in the Golgi complex could impair both protein transport-and non-transport-associated functions (for instance, protein and lipid glycosylation) [Axelsson et al, 2001;Kellokumpu et al, 2002]. In line with this hypothesis, interference with either the activity of (V)H þ -ATPase [Yilla et al, 1993] or with actin polymerization [Hirschberg et al, 1998;Jacob et al, 2003;Cobbold et al, 2004] results in an altered post-Golgi transport. Consistently with these observations, there is an increase in the surface membrane area of the Golgi (Table I) and an abnormal accumulation of peri-Golgi transport carriers in actin toxinstreated cells (Figs.…”

Influenza infection modulates vesicular trafficking and induces Golgi complex disruption

“…Normally VSV-G is co-translationally translocated into the ER and, within few minutes exits the ER and transported to the Golgi through the ERGIC [15]. However, VSVG-ts045 is misfolded at 40°C in ER-like structures.…”

Extracellular wildtype and mutant SOD1 induces ER–Golgi pathology characteristic of amyotrophic lateral sclerosis in neuronal cells

“…Vesicular stomatitis virus glycoprotein ts045 (VSVG-ts045) is a widely employed marker used to examine eR-Golgi transport [35]. VSVG-ts045 misfolds and is retained in the eR at 40 °C, but is transported to the Golgi apparatus at the permissive temperature of 32 °C [35].…”

“…Vesicular stomatitis virus glycoprotein ts045 (VSVG-ts045) is a widely employed marker used to examine eR-Golgi transport [35]. VSVG-ts045 misfolds and is retained in the eR at 40 °C, but is transported to the Golgi apparatus at the permissive temperature of 32 °C [35]. We first examined eR-Golgi transport in Sh-SY5Y cells transfected with SOD1 proteins linked to eGFP [6,12] and VSVG-ts045 mCherry, to confirm that intracellular mutant SOD1 can inhibit eR-Golgi trafficking in this cell line.…”