Related Experiment Video
Updated: Jul 21, 2026
Mitochondria-associated ER Membranes (MAMs) and Glycosphingolipid Enriched Microdomains (GEMs): Isolation from Mouse Brain
Published on: March 4, 2013
Sphingomyelin-enriched microdomains at the Golgi complex
I Gkantiragas1, B Brügger, E Stüven
1Biochemie-Zentrum Heidelberg (BZH), University of Heidelberg, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany.
Abstract:
Sphingomyelin- and cholesterol-enriched microdomains can be isolated as detergent-resistant membranes from total cell extracts (total-DRM). It is generally believed that this total-DRM represents microdomains of the plasma membrane. Here we describe the purification and detailed characterization of microdomains from Golgi membranes. These Golgi-derived detergent-insoluble complexes (GICs) have a low buoyant density and are highly enriched in lipids, containing 25% of total Golgi phospholipids including 67% of Golgi-derived sphingomyelin, and 43% of Golgi-derived cholesterol. In contrast to total-DRM, GICs contain only 10 major proteins, present in nearly stoichiometric amounts, including the alpha- and beta-subunits of heterotrimeric G proteins, flotillin-1, caveolin, and subunits of the vacuolar ATPase. Morphological data show a brefeldin A-sensitive and temperature-sensitive localization to the Golgi complex. Strikingly, the stability of GICs does not depend on its membrane environment, because, after addition of brefeldin A to cells, GICs can be isolated from a fused Golgi-endoplasmic reticulum organelle. This indicates that GIC microdomains are not in a dynamic equilibrium with neighboring membrane proteins and lipids. After disruption of the microdomains by cholesterol extraction with cyclodextrin, a subcomplex of several GIC proteins including the B-subunit of the vacuolar ATPase, flotillin-1, caveolin, and p17 could still be isolated by immunoprecipitation. This indicates that several of the identified GIC proteins localize to the same microdomains and that the microdomain scaffold is not required for protein interactions between these GIC proteins but instead might modulate their affinity.
Insights
Researchers purified Golgi-derived detergent-insoluble complexes (GICs), revealing stable lipid-protein microdomains distinct from plasma membrane domains. These GICs maintain integrity even after organelle fusion, suggesting a non-dynamic scaffold.
Area of Science:
- Cell Biology
- Membrane Biology
- Biochemistry
Background:
- Lipid- and cholesterol-enriched microdomains, known as detergent-resistant membranes (DRMs), are typically isolated from total cell extracts and presumed to originate from the plasma membrane.
- Understanding the precise origin and characteristics of these microdomains is crucial for elucidating cellular signaling and membrane trafficking pathways.
Purpose of the Study:
- To purify and characterize microdomains originating from Golgi membranes.
- To investigate the lipid and protein composition of these Golgi-derived microdomains.
- To determine the stability and dynamic properties of these unique membrane structures.
Main Methods:
- Isolation of Golgi-derived detergent-insoluble complexes (GICs) from cell extracts.
- Biochemical analysis of lipid and protein content within GICs.
- Morphological studies using brefeldin A treatment to assess localization and stability.
- Cholesterol extraction experiments followed by immunoprecipitation to analyze protein interactions.
Main Results:
- GICs were successfully purified from Golgi membranes, exhibiting low buoyant density and enrichment in sphingomyelin and cholesterol.
- GICs contained a distinct set of approximately 10 major proteins, including G protein subunits, flotillin-1, caveolin, and vacuolar ATPase subunits.
- GIC stability was independent of the membrane environment, persisting even after Golgi-ER fusion induced by brefeldin A.
- Disruption of GICs by cholesterol depletion still allowed for the isolation of protein subcomplexes, indicating stable protein-protein interactions within the microdomain.
Conclusions:
- Golgi membranes contain distinct, stable detergent-insoluble microdomains (GICs) separate from plasma membrane DRMs.
- The stability of GICs suggests they are not in dynamic equilibrium with surrounding membrane components.
- The identified GIC proteins interact within these microdomains, with the scaffold potentially modulating their functional affinities rather than mediating initial interactions.
Related Concept Videos
01:49Golgi Apparatus
01:18Membrane Domains
Protein Domains
The membrane comprises a group of distinct proteins responsible for carrying out a cell's specific function. For example, the plasma membrane of the human sperm, or a single germ cell, contains a unique set of proteins in the anterior...
01:38Intralumenal Vesicles and Multivesicular Bodies
01:09Golgi Apparatus
The Golgi apparatus is a eukaryotic organelle that has a distinctive ribbon-like appearance. It is a primary sorting and dispatch station for cargo arriving from the ER. Newly arriving vesicles enter the cis face of the Golgi, closest to the ER, and are...
01:12Golgi Matrix Proteins
One of the first identified Golgi matrix proteins was GM130, a rod-like protein located in the cis-Golgi. Subsequently, many Golgi...
01:09Golgi Apparatus
The Golgi apparatus is a eukaryotic organelle that has a distinctive ribbon-like appearance. It is a primary sorting and dispatch station for cargo arriving from the ER. Newly arriving vesicles enter the cis face of the Golgi, closest to the ER, and are...