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Reconstituting membrane flow
T I B S - March 1985
95
Reconstituting membrane flow Eukaryotic cells contain many different compartments whose surrounding membranes are chemically and functionally distinct. How is this diversity generated'? All membranes of a eukaryotic cell (except those of mitochondria, chloroplasts and perhaps also peroxisomes) originate from the endoplasmic reticulum and acquire their unique properties by a complex differentiation process in which vesicles bud off from the endoplasmic reticulum and fuse with cisternae of the Golgi complex. The Golgi complex, which occupies a central role in the 'sorting' of membrane components, then distributes membrane material to the plasma membrane, tysosomes, secretory granules and perhaps also the nuclear envelope. This distribution is probably also mediated by vesicles that bud from dilated ends of the Golgi cisternae and fuse with the appropriate acceptor membrane. Most steps of this intracellular 'membrane flow' are accompanied by the removal of a select subpopulation of membrane components and the covalent modification of membrane proteins. For example, the endoplasmic reticulum and the individual Golgi cisternae contain unique enzymes that participate in the ordered and sequential addition of sugar groups to amino acid residues in proteins. 'Coreglycosylation' occurs in the endoplasmic reticulum; removal of mannose residues in Golgi cisternae facing the endoplasmic reticulum ('cis-Golgi'); addition of N-acetylglucosamine (GIcNAc) in 'medial' Golgi cisternae; and addition of galactose residues in Golgi cisternae facing the plasma membrane ('trarL~Golgi'). The sugar branches of a glycoprotein are, thus, a molecular record of the intracellular compartments to which the protein had been exposed. What are the mechanisms that regulate membrane flow and endow it with such exquisite specificity? At first slght it seems almost hopeless to study the interaction between different cell organelles by biochemical methods since these methods usually require homogenization of cells, purification of components and reconstitution of a process in vitro. However, in a remarkable series of papers, Rothman and his colleagues have recently described the reconstitution of membrane flow between cisternae of isolated Golgi complexes ~ 3. How could they reconstitute a process that at first sight seems to be inherently non-
reconstitutable? They measured membrane flow by a rapid and sensitive complementation assay based on the covalent addition of [3H]-GIcNAc to the viral-eiacoded membrane glycoprotein of the vesicular stomatitis virus (VSV Gprotein). As a 'donor' compartment they used Golgi complexes purified from the VSV-infected Chinese hamster ovary (CHO) cell line 15B. This mutant cell line lacks GIcNAc transferase I and is, thus, unable to attach [3H]-GIcNAc to core-glycosylated G-protein. As an 'acceptor' compartment they used either Golgi fractions from uninfected, wildtype CHO cells or from rat liver. In this system, transfer of the incompletely glycosylated G-protein from the donorto the acceptor-cisternae could b e detected by the attachment of [3H]GicNAc to G-protein. Such a transfer was indeed found. Surprisingly, rough calculations suggested that transfer of G-protein between cisternae of the artificially mixed Golgi stacks was nearly as efficient as transfer in vivo. The combination of a sensitive assay with a complex experimental system always invites artefacts; Rothman and his colleagues, therefore, present many controls to show that their results indeed reflect inter-Golgi transfer of VSV G-protein. Non-specific fusion between different membranes was shown to be proved unlikely by demonstrating that the donor- and acceptor-Golgi complexes remained morphologically distinct. Also, transfer in vitro required ATP and cytosolic proteins and was blocked by N-ethylmaleimide. Sequential incubations in the presence or absence of ATP, cytosolic proteins or N-ethylmaleimide showed that the in vitro transfer proceeded in at least three steps: (1) 'priming' of the donor membranes (allowing them to donate their G-protein without a lag); (2) transfer of G-protein to the acceptor membranes; and (3) delivery of G-protein to the functional GIcNAc transferase 1 inside the medial cisternae of the wild-type acceptor Golgi complex. To show that their data did not merely reflect entry of a few GlcNAc transferase I molecules from the acceptor- to the donor-cisternae, the authors used Golgi complexes from rat liver as acceptor membranes; since these are morphologically different from CHO Golgi complexes, electron microscope autoradiography could be used to show that glycoprotein labeled with [3H]-GIcNAc was, in fact, located in the cisternae of the acceptor (but not
those of the donor) Golgi complexes. A striking series of electron micrographs in the third paper of this series suggests that the 'priming' reaction mentioned above may correspond to coating (with clathrin molecules?) of the ends of Golgi cisternae and the subsequent pinching-off of coated vesicles. These intermediate structures are reminiscent of the clathrin-coated, Golgirelated vesicles recently described by Orci and his colleagues in insulinproducing pancreatic B-cells4. These exciting developments present biochemists with a plethora of specific questions: How do ATP and cytosolic proteins induce the attachment of clathrin to Golgi membranes? Are there additional proteins required for triggering fusion of the donor vesicles to the acceptor membranes? What are the receptors ensuring the specificity of these interactions? Are these events accompanied by transient covalent modification of clathrin molecules? Since all these questions can be tested experimentally, we should soon know a good deal more about how membranes flow in living cells. References 1 Balch, W. E., Dunphy. W. G., Braell. W. A. and Rothman, J. E. (1984) ('ell 39, 41~416 2 Braell, W. A., Balch, W. E., Dobbertin, D. C. and Rothman, J. E. (1984) Cell 39, 511-524 3 Balch, W. E., Glick. B. S. and Rothman, J. E. (1984) Cell 39, 525-536 4 Orci, L., Halban, P., Amherdt, M., Ravazzola, M., Vasalli. J.-D. and Perrelet, A. (1984) Cell 39, 39-47 GO'ITFRIED SCHATZ Biocenter, University of Basel, CH-4056 Basel, Switzerland.
© 1985.ElsevierSciencePublishersB,V,, Amsterdam (1376 5067/85~$112(10
95
Reconstituting membrane flow Eukaryotic cells contain many different compartments whose surrounding membranes are chemically and functionally distinct. How is this diversity generated'? All membranes of a eukaryotic cell (except those of mitochondria, chloroplasts and perhaps also peroxisomes) originate from the endoplasmic reticulum and acquire their unique properties by a complex differentiation process in which vesicles bud off from the endoplasmic reticulum and fuse with cisternae of the Golgi complex. The Golgi complex, which occupies a central role in the 'sorting' of membrane components, then distributes membrane material to the plasma membrane, tysosomes, secretory granules and perhaps also the nuclear envelope. This distribution is probably also mediated by vesicles that bud from dilated ends of the Golgi cisternae and fuse with the appropriate acceptor membrane. Most steps of this intracellular 'membrane flow' are accompanied by the removal of a select subpopulation of membrane components and the covalent modification of membrane proteins. For example, the endoplasmic reticulum and the individual Golgi cisternae contain unique enzymes that participate in the ordered and sequential addition of sugar groups to amino acid residues in proteins. 'Coreglycosylation' occurs in the endoplasmic reticulum; removal of mannose residues in Golgi cisternae facing the endoplasmic reticulum ('cis-Golgi'); addition of N-acetylglucosamine (GIcNAc) in 'medial' Golgi cisternae; and addition of galactose residues in Golgi cisternae facing the plasma membrane ('trarL~Golgi'). The sugar branches of a glycoprotein are, thus, a molecular record of the intracellular compartments to which the protein had been exposed. What are the mechanisms that regulate membrane flow and endow it with such exquisite specificity? At first slght it seems almost hopeless to study the interaction between different cell organelles by biochemical methods since these methods usually require homogenization of cells, purification of components and reconstitution of a process in vitro. However, in a remarkable series of papers, Rothman and his colleagues have recently described the reconstitution of membrane flow between cisternae of isolated Golgi complexes ~ 3. How could they reconstitute a process that at first sight seems to be inherently non-
reconstitutable? They measured membrane flow by a rapid and sensitive complementation assay based on the covalent addition of [3H]-GIcNAc to the viral-eiacoded membrane glycoprotein of the vesicular stomatitis virus (VSV Gprotein). As a 'donor' compartment they used Golgi complexes purified from the VSV-infected Chinese hamster ovary (CHO) cell line 15B. This mutant cell line lacks GIcNAc transferase I and is, thus, unable to attach [3H]-GIcNAc to core-glycosylated G-protein. As an 'acceptor' compartment they used either Golgi fractions from uninfected, wildtype CHO cells or from rat liver. In this system, transfer of the incompletely glycosylated G-protein from the donorto the acceptor-cisternae could b e detected by the attachment of [3H]GicNAc to G-protein. Such a transfer was indeed found. Surprisingly, rough calculations suggested that transfer of G-protein between cisternae of the artificially mixed Golgi stacks was nearly as efficient as transfer in vivo. The combination of a sensitive assay with a complex experimental system always invites artefacts; Rothman and his colleagues, therefore, present many controls to show that their results indeed reflect inter-Golgi transfer of VSV G-protein. Non-specific fusion between different membranes was shown to be proved unlikely by demonstrating that the donor- and acceptor-Golgi complexes remained morphologically distinct. Also, transfer in vitro required ATP and cytosolic proteins and was blocked by N-ethylmaleimide. Sequential incubations in the presence or absence of ATP, cytosolic proteins or N-ethylmaleimide showed that the in vitro transfer proceeded in at least three steps: (1) 'priming' of the donor membranes (allowing them to donate their G-protein without a lag); (2) transfer of G-protein to the acceptor membranes; and (3) delivery of G-protein to the functional GIcNAc transferase 1 inside the medial cisternae of the wild-type acceptor Golgi complex. To show that their data did not merely reflect entry of a few GlcNAc transferase I molecules from the acceptor- to the donor-cisternae, the authors used Golgi complexes from rat liver as acceptor membranes; since these are morphologically different from CHO Golgi complexes, electron microscope autoradiography could be used to show that glycoprotein labeled with [3H]-GIcNAc was, in fact, located in the cisternae of the acceptor (but not
those of the donor) Golgi complexes. A striking series of electron micrographs in the third paper of this series suggests that the 'priming' reaction mentioned above may correspond to coating (with clathrin molecules?) of the ends of Golgi cisternae and the subsequent pinching-off of coated vesicles. These intermediate structures are reminiscent of the clathrin-coated, Golgirelated vesicles recently described by Orci and his colleagues in insulinproducing pancreatic B-cells4. These exciting developments present biochemists with a plethora of specific questions: How do ATP and cytosolic proteins induce the attachment of clathrin to Golgi membranes? Are there additional proteins required for triggering fusion of the donor vesicles to the acceptor membranes? What are the receptors ensuring the specificity of these interactions? Are these events accompanied by transient covalent modification of clathrin molecules? Since all these questions can be tested experimentally, we should soon know a good deal more about how membranes flow in living cells. References 1 Balch, W. E., Dunphy. W. G., Braell. W. A. and Rothman, J. E. (1984) ('ell 39, 41~416 2 Braell, W. A., Balch, W. E., Dobbertin, D. C. and Rothman, J. E. (1984) Cell 39, 511-524 3 Balch, W. E., Glick. B. S. and Rothman, J. E. (1984) Cell 39, 525-536 4 Orci, L., Halban, P., Amherdt, M., Ravazzola, M., Vasalli. J.-D. and Perrelet, A. (1984) Cell 39, 39-47 GO'ITFRIED SCHATZ Biocenter, University of Basel, CH-4056 Basel, Switzerland.
© 1985.ElsevierSciencePublishersB,V,, Amsterdam (1376 5067/85~$112(10