Golgi Isolation

Abstract

The Golgi apparatus is a membranous organelle that modifies and packages proteins and lipids into transport carriers and sends them to the proper locations in the cell. The study of Golgi structure and function can be facilitated by the isolation of this organelle from homogenates of tissues or cells. Liver cells have abundant Golgi membranes because they actively secrete proteins and lipids; therefore, liver tissue is often the preferred source. In this protocol, Golgi membranes are purified from rat liver homogenate by two sequential sucrose gradients. The relative yield of the prepared Golgi stacks is then assessed by measuring the increase in activity of a Golgi marker enzyme, β-1,4-galactosyltransferase, over that of the total liver homogenate. A typical preparation can yield Golgi membranes that are purified 80- to 100-fold over the homogenate, and the majority (60%-70%) retain their stacked nature.

GRASP55 Senses Glucose Deprivation through O-GlcNAcylation to Promote Autophagosome-Lysosome Fusion

“…Lysate was cleared by centrifugation, incubated with indicated antibodies overnight at 4 C, subsequently incubated with protein A beads for another 2 h, reisolated and blotted with O-GlcNAc antibodies, CTD110.6 or RL2. To determine whether phosphorylation of GRASP55 in mitosis affects its O-GlcNAcylation, Rat Liver Golgi (RLG) membranes (Tang and Wang, 2015) were first incubated with interphase cytosol (IC) or mitotic cytosol (MC) for 1 h at 37 C, then recovered by ultracentrifugation in a TLA55 rotor at 55,000 rpm and 4 C for 1 h. The membrane pellets were lysed in 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Triton X-100 and protease inhibitors and subjected to immunoprecipitation with a rat GRASP55 antibody. Rat liver Golgi membranes, and cytosols were prepared as previously described (Rabouille et al, 1995;Tang et al, 2010a).…”

Mitotic phosphorylation inhibits the Golgi mannosidase MAN1A1

“…To identify candidate proteins regulated by phosphorylation during the cell cycle, we purified interphase Golgi membranes from rat liver (RLG), prepared mitotic Golgi fragments (MGFs) by incubating RLG with mitotic cytosol ( Tang and Wang, 2015 ; Wang et al, 2006 ; Tang et al, 2010 ), and performed mass spectrometry (MS) analysis to identify phosphorylated proteins ( Kweon and Andrews, 2013 ). The results revealed previously reported mitotic phosphorylation of Golgi structure proteins, including GM130 ( Lowe et al, 2000 ), GRASP65 ( Tang et al, 2012 ), Giantin ( Dephoure et al, 2008 ; Olsen et al, 2010 ), and Golgin 84 ( Diao et al, 2003 ; Dephoure et al, 2008 ) ( Table S1 ), as well as a number of Golgi enzymes, including MAN1A1, MAN1A2, ST6GAL1, and GALNT11 ( Figure 1A ; Table S1 ).…”

Sulfoproteomics Workflow with Precursor Ion Accurate Mass Shift Analysis Reveals Novel Tyrosine Sulfoproteins in the Golgi

“…The Golgi apparatus was isolated from rat liver by a sucrose gradient . Mitotic rat Golgi membranes were prepared by treating with the mitotic HeLa cytosol in vitro. − Golgi preparations were reconstituted in 8 M urea with a complete EDTA-free protease inhibitor mixture (Roche) and Pierce phosphatase inhibitor mix (Thermo Scientific) using a microprobe sonicator on ice.…”