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Vesicular transport in cells – from analysis to biogenesis
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.
Camillo Golgi (1843-1926). © Universitá degli studi di Pavia
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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).
Three types of transport containers
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 transGolgi 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.
Vesicular transport in a cell involving endocytotic and exocytotic vesicles © Nature Rev. Mol. Cell Biol. 10, 360-364
(2009)
The group of researchers led by Prof. Dr. Felix T. Wieland at the Biochemistry Centre at the
University of Heidelberg (BZH) has been carrying out an intensive investigation of the structure,
function and biogenesis of COPI vesicles. In cooperation with a group of researchers led by Prof.
James E. Rothman (then at the Sloan Kettering Institute in New York, now at Yale University),
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Wieland’s team isolated and purified a quantity of COPI vesicles that was large enough to
enable the chemical analysis of the COPI components. They made use of Rothman’s
groundbreaking discovery that the isolated Golgi apparatus is able to form COPI vesicles in
vitro [editor’s note: Ways to isolate the Golgi apparatus itself, using classical cell fractionation
methods including ultracentrifugation in sugar density gradients, were developed as early as
the end of the 1960s, amongst others by J. D. Morré at Purdue University, West Lafayette,
Indiana, with the participation of the author of this article.]
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.
Biogenesis of COPI vesicles
Prof. Dr. Felix T. Wieland, Heidelberg University Biochemistry Centre © University of Heidelberg
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 membranebound 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
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composition of the membrane also plays a role in the formation of vesicles.
The lipids of the COPI 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.
Transmission electron microscope image of a Golgi apparatus (composed of membrane-bound stacks of cisternae)
and secretory vesicles. © Institute of Cell Biology, University of Freiburg
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.
Further information:
Prof. Dr. Felix Wieland
Biochemistry Centre at the University of Heidelberg (BZH)
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E-mail: felix.wieland(at)bzh.uni-heidelberg.de
PD Dr. Britta Brügger
Biochemistry Centre at the University of Heidelberg (BZH)
E-mail: britta.bruegger(at)bzh.uni-heidelberg.de
Article
12-Jul-2010
EJ
BioRN
© BIOPRO Baden-Württemberg GmbH
The article is part of the following dossiers
Membrane proteins
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