Cell Biology

Protein sorting at the trans Golgi network (TGN) plays important roles in targeting newly synthesized proteins to their specific destinations. The aim of this proposal is to reconstitute the packaging of non-Golgi resident cargo proteins into vesicles at the TGN, utilizing rat liver cytosol, semi-intact mammalian cells and nucleotides. The protocol describes how to perform the vesicle formation assay, how to isolate vesicles and how to detect cargo proteins in vesicles. This reconstitution assay can be used to quantitatively measure the efficiency of the packaging of a specific cargo protein into transport vesicles at the TGN under specific experimental conditions.

Intra-Golgi retrograde vesicle transport is used to traffic and sort resident Golgi enzymes to their appropriate cisternal locations. An assay was established to investigate the molecular details of vesicle targeting in a cell-free system. Stable cell lines were generated in which the trans-Golgi enzyme galactosyltransferase (GalT) was tagged with either CFP or YFP. Given that GalT is recycled to the cisterna where it is located at steady state, GalT-containing vesicles target GalT-containing cisternal membranes. Golgi membranes were therefore isolated from GalT-CFP expressing cells, while vesicles were prepared from GalT-YFP expressing ones. Incubating CFP-labelled Golgi with YFP-labelled vesicles in the presence of cytosol and an energy regeneration mixture at 37 °C produced a significant increase in CFP-YFP co-localization upon fluorescent imaging of the mixture compared to incubation on ice. The assay was validated to require energy, proteins and physiologically important trafficking components such as Rab GTPases and the conserved oligomeric Golgi tethering complex. This assay is useful for the investigation of both physiological and pathological changes that affect the Golgi trafficking machinery, in particular, vesicle tethering.

This protocol details the isolation of enriched Golgi membranes from rat liver, using discontinuous density gradient centrifugation. This high-yield extraction method is useful for several applications, including immunoprecipitation of solubilised Golgi membrane proteins (preparation included) and electron microscopy. Protocol adapted from Leelavathi et al. (1970).

This is a protocol to extract intact Golgi Membranes from HeLa cells using sucrose gradient centrifugation. This extraction is very useful for several applications including pull-down of Golgi membrane proteins, electron microscopy and reconstitution of protein transport into an isolated system. Protocol adapted from Balch et al. (1984).

This protocol describes how to separate the endoplasmic reticulum (ER) and Golgi apparatus in yeast cells using a subcellular fractionation approach with an Accudenz gradient.