COPI vesicles, one of the three major types of intracellular transport vesicles, are an excellent example of how the molecular analysis of individual vesicle components can lead to an understanding of the biogenesis and transport mechanisms of membrane vesicles as a whole. The research carried out by Dr. Felix T. Wieland’s team at the University of Heidelberg has made a decisive contribution to such detailed insights.
Eukaryotic cells are characterised by complex membrane systems that separate different metabolic compartments from each other at the same time as transporting metabolic products and messenger substances to specific destinations inside the cell and into the extracellular space. The transport is mediated by vesicles that detach from the folded and branched cisternae of intracellular membranes. They either then fuse with intracellular membranes again or with the plasma membrane where the contents of the vesicles, secretory proteins for example, are discharged to the outside of the cell. This process is referred to as exocytosis. The opposite process is known as endocytosis, in which cells take up molecules from the exterior by enveloping them in their cell membrane before releasing them inside the cell.
The Golgi apparatus, named after the Italian physiologist Camillo Golgi who discovered a method to stain nervous tissue with silver, which led to his discovery in 1898 of the "apparato reticulare interno" in the hippocampal neurons, is integral in modifying and packaging macromolecules for exocytosis or use within the cell (endocytosis). Camillo Golgi was awarded the Nobel Prize in Physiology and Medicine in 1906 for his work on the structure of the nervous system. However, the importance of the Golgi apparatus and the intracellular membrane system was recognised much later, in particular through the research of Albert Claude, Christian de Duve and George Palade (Nobel Prize 1974) and Günter Blobel (Nobel Prize 1999).
The mechanism of intracellular vesicular transport has since turned out to be quite complex: molecules are transported into and outside of cells, a process for which different cargo containers are used. Three major types of transport vesicles have been analysed in detail on the molecular level, and are defined through the protein coats surrounding their membranes. The first type of transport vesicles known was the so-called “clathrin coated vesicles” (CVV), which bud into the cytoplasm (endocytosis) and also mediate the transport between the trans-Golgi network, plasma membrane, endosomes and lysosomes. The two other types of transport vesicles, which are known as COPI- and COPII vesicles (COP stands for ‘coat protein’), are responsible for intracellular cargo transport: COPII vesicles transport newly synthesised secretory and membrane-bound proteins from the endoplasmic reticulum (ER) to the Golgi apparatus, or more precisely, to the so-called ‘intermediate compartment’ located between the ER and the Golgi apparatus. COPI vesicles mediate the transport inside the Golgi apparatus, i.e. between the individual cisternae of the Golgi apparatus, as well as the retrograde transport of cargo from the cis-Golgi network to the ER.
Wieland and his team showed that the COPI vesicles form at the Golgi apparatus by binding a soluble cytosolic coat protein, which forms a complex of seven subunits (COPs) that is referred to as coatomer. Genome sequencing revealed that two (gamma and zeta) of the seven subunits occur as isotypes. Different COP combinations lead to three coatomer isotypes, stoichiometrically composed of seven subunits, namely gamma1/zeta1, gamma1/zeta2 and gamma2/zeta1. These three coatomer complexes are located at different sites in the vesicular transport system and are thought to have different functions. Researchers were able to isolate vesicles containing the gamma2/zeta1 coatomer isotype from the other COPI vesicles.
Another cytosolic component required for the budding of COPI vesicles at the Golgi apparatus is the ADP ribosylation factor 1 (Arf1), a small GTP-binding protein. Stimulated by a membrane-bound GTPase-activating protein (GAP), GTP is hydrolysed to GDP by ARF during vesicle formation. The membrane also contains transmembrane proteins from the p24 protein family which act as coat receptors. The Heidelberg researchers have developed a detailed model on the step-wise budding of COPI vesicles from the Golgi membrane based on the molecular characteristics and the binding behaviour of the individual components. This model was tested in vitro in a liposomal system and, amongst other things, has been used to explain that coatomer is bound to the Golgi apparatus via ARF and p24 proteins. This leads to a conformational change of coatomer, which in turn leads to its polymerisation on the membrane and makes the membrane bend into a bud shape. It is assumed that the lipid composition of the membrane also plays a role in the formation of vesicles.
It has been known for many decades that each cell organelle has a typical lipid composition. Since the organelles, for example ER and Golgi apparatus, are connected with each other by way of vesicular membrane transport, one could perhaps assume that the lipid composition of the transport vesicles corresponds to that of the donor or recipient membrane or to an intermediary pattern of the two. When the Heidelberg team analysed the lipids of the COPI vesicles and those of their donor membranes, i.e. the Golgi cisternae, they found that the vesicles had a sphingomyelin and cholesterol (two lipids that are found in very high concentrations in the plasma membrane) content that was considerably lower than that of the Golgi membranes.
The researchers were particularly surprised to find that the concentration of a certain sphingomyelin species, which contains the fatty acid stearic acid (18:0), was 12 times higher in COPI vesicles than in the Golgi membrane. The researchers concluded that the vesicular membrane proteins specifically interact with the lipids, which leads to their selection and sorting.
Many questions relating to the biogenesis and the transport of COPI vesicles are of course not yet solved. In two projects funded by the DFG as part of the cooperative research centre "Dynamics of macromolecular complexes involved in biosynthetic transport" (SFB 638), Wieland is currently focusing on the "Dynamics of COPI assembly" (in cooperation with PD Dr. Britta Brügger from BZH) and the "Dynamics of coatomer structures" (in cooperation with Dr. John Briggs from the European Molecular Biology Laboratory in Heidelberg). Wieland is the spokesperson of the SFB 638 which went into its second funding period in January 2008.