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Vacuolar Sorting: Tracking down an elusive receptor
ROWAN E. CHAPMAN
VACUOLAR SORTING
Tracking down an elusive receptor The receptor that sorts carboxypeptidase Y for transport to the vacuole in yeast has recently been identified; how vacuolar sorting is regulated is far from understood, but an intriguing model has been proposed. Understanding the complex organization and operation of the eukaryotic secretary pathway is an exciting challenge for cell biologists. How does transport occur between membrane-bound compartments within cells? And given the continuous flux between these compartments, how do they retain their distinct composition, and thus their identity? We now have a good idea about the routes proteins take through the secretary pathway, and about the amino-acid 'signal' sequences required for residence in most of the compartments. However, little is known about the actual sorting mechanism by which proteins carrying the appropriate signal sequences are recognized and directed to the appropriate compartment. The recent identification of the sorting receptor for the vacuolar protein carboxypeptidase Y in yeast [1] suggests that this state of ignorance may soon be a thing of the past. In both yeast and mammalian cells, sorting decisions must be made at every step along the secretary pathway (Fig. 1). According to the widely accepted 'bulk flow' hypothesis, no specific information is required for proteins to progress along the pathway to secretion from the cell. There must, therefore, be positive signals that prevent proteins destined for residence in a compartment along the pathway, or one reached by a branch off the
main pathway, from moving forward with the bulk flow. The first compartment in the pathway is the endoplasmic reticulum (ER), where proteins that take the secretary pathway are synthesized and folded, and where their glycosylation begins. The proteins then move to the Golgi apparatus, a series of membranous subcompartments in which further processing reactions - glycosylation, lipid modification and proteolysis - occur. The Golgi apparatus is where the first sorting decision is made, in which ER-resident proteins are separated from the rest and directed - presumably along with ER-specific lipids - back to the ER. The retention of ER-resident proteins thus operates via a retrieval mechanism, rather than by inhibition of their forward transport (reviewed in [2]). Specific amino-acid sequences that are necessary and sufficient for proteins to be retrieved in this way have been identified in both soluble and integral membrane ER proteins (the signals being, in the singleletter code, H/KDEL and KKXX, respectively) [2]. The retention of Golgi-resident glycosyltransferases, on the other hand, does not appear to rely on specific aminoacid sequences, depending rather on something about the length and composition of their transmembrane domains that has not yet been precisely defined [3]; as
Fig. 1. The eukaryotic secretory pathway. Movement between the different organelles is widely thought to occur via carrier vesicles. The yeast vacuole and mammalian lysosome share many features and are though to represent functionally equivalent compartments; both contain most of the cells' hydrolytic enzymes, have an acidic interior and are derived from a branch of the secretory pathway. Complete arrows indicate experimentally established transport steps; broken arrows indicate predicted, but not yet established, steps.
© Current Biology 1994, Vol 4 No 11
1019
1020
Current Biology 1994, Vol 4 No 11 yet there is no evidence that these enzymes are retained within the Golgi apparatus in a similar manner to the ER-resident proteins (that is, by a recycling mechanism). After passage through, and modification within, subcompartments of the Golgi apparatus, proteins move to the trans-Golgi network, a specialized processing and sorting compartment. Proteins resident in this compartment appear (like ER-resident proteins) to have specific amino-acid sequences that direct their retrieval from a post-Golgi compartment [2]. For those proteins destined to move on, the pathway bifurcates; they are either sorted to the lysosomal/vacuolar system or continue on the route to the plasma membrane. In both yeast and mammalian cells, specific signal sequences have been identified that are sufficient to direct soluble hydrolases to the lysosome/vacuole (reviewed in [4,5]). The situation is slightly more complicated in the case of integral membrane proteins; in mammalian cells, integral membrane proteins lacking a specific signal sequence accumulate at the plasma membrane, whereas in yeast they end up in the vacuole. However this apparent difference in 'default' localization is not completely clear-cut, as in mutant yeast cells with defects of the clathrin heavy chain, the trans-Golgi protein Kex2p is mislocalized to the plasma membrane rather than to the vacuole [6]. Further work is needed to sort out exactly what is going on. Despite this, many aspects of the secretory pathway compartments and sorting signals seem to be remarkably well conserved throughout the eukaryote kingdom. Much less is known about the molecular mechanisms involved in recognizing these compartmental 'address labels'. Until recently, only two receptor molecules that sort proteins away from the bulk flow within the pathway had been identified: erd2, the recycling receptor that recognizes the H/KDEL amino-acid signal on soluble ERresident proteins [2], and the mannose-6-phosphate receptor, which recognizes a specific sugar-phosphate linkage attached to a subset of mammalian hydrolases and directs the recognized enzymes to the lysosome [5]. Now, a third receptor has been identified in yeast [1]; this is a molecule that specifically recognizes the yeast enzyme carboxypeptidase Y and directs it to the vacuole. The identification of the carboxypeptidase Y receptor comes ten years after the initial genetic screens for yeast mutants defective in vacuolar protein sorting (known as vps mutants). The screens were set up to look for mutants that secrete the incompletely processed, Golgi form of carboxypeptidase Y (p2CPY), and are thus defective in sorting proteins from the trans-Golgi to the vacuole. The vps mutations identified in several screens were grouped into 45 complementation groups [7]. Many of these were found to be allelic with genes identified in screens for mutants with altered vacuolar morphology, Ca2 +-resistance and sensitivity to osmotic stress. Further analysis showed that as well as their p2CPY-secretion defect, most
of the mutants are defective in sorting the soluble vacuolar proteins proteinase A and proteinase B, although vacuolar membrane proteins seemed to stay inside the mutant cells. These pleiotropic phenotypes, along with the sheer number of complementation groups, indicate that many proteins are required for correct targeting of soluble vacuolar proteins (reviewed in [8]). Despite this complexity, there was evidence suggesting that amongst the VPS genes there would be one encoding a specific receptor for carboxypeptidase Y. Firstly, the carboxypeptidase-Y-sorting mechanism was shown to be saturable - over-expression of carboxypeptidase Y in wild-type cells results in its secretion from the cell. This is a specific effect, as proteinase A secretion does not result from over-expression of carboxypeptidase A, nor .does over-expression of proteinase A cause carboxypeptidase Y secretion. Sorting of carboxypeptidase Y relies on a specific amino-acid sequence (QRPL) at its amino terminus; changing this to KRPL abolishes sorting to the vacuole. With this information, Scott Emr and colleagues set out to find the carboxypeptidase Y receptor gene amongst the mass of carboxypeptidase-Y-secreting vps mutants. They found three mutant yeast strains, vpslO, vps35 and vps29, that appeared specifically to missort carboxypeptidase Y and to have no other defects [9]. Three genes were thus implicated in carboxypeptidase Y sorting, and the first of these to be cloned was VPS35. Despite the specificity of the carboxypeptidase-Y-sorting defect in the vps35 mutant, it was hard to see how VPS35 could encode the carboxypeptidase Y receptor as the gene product product, Vps35p, is peripherally associated with the cytoplasmic face of Golgi membranes [9], whereas the precursor form of carboxypeptidase Y, p2CPY, is a lumenal protein. Cloning of VPSIO was a different story. The recent paper from Emr and colleagues [1] shows quite clearly that VpslOp has the appropriate structure, intracellular location and p2CPYbinding properties to be a specific carboxypeptidase-Ysorting receptor. The large, 1413-residue VpslOp is an integral membrane protein located in the trans-Golgi sorting compartment. Furthermore, it has a large lumenal domain that can be specifically cross-linked to p2CPY. This interaction is dependant on the carboxypeptidase Y sorting signal, QRPL, as a sorting-deficient form (with QRPL changed to KRPL) cannot be cross-linked to p2CPY under the same conditions. The QRPL signal is sufficient to direct the association of a reporter molecule with VpslOp. Taken together, these results provide compelling evidence that VpslOp is a carboxypeptidase Y receptor. It seems unlikely, however, that VpslOp is a receptor exclusively for carboxypeptidase Y, as it can be specifically cross-linked to two further proteins, which could be hitherto unidentified VpslOp ligands. VpslOp is probably one member of a family of hydrolase receptors that includes sorting-receptors for proteinases A and B. These are unlikely to be encoded by any of the VPS genes, as
DISPATCH
there are no vps mutants that specifically missort either proteinase whilst retaining carboxypeptidase Y, the predicted phenotype of a mutation in a proteinase A or proteinase B receptor. Additional screens will therefore be required to identify further vacuolar-protein-sorting receptors. The occurrence in yeast of more than one vacuolar-targeting signal, and thus more than one sorting receptor, is similar to the situation in mammalian cells, where both mannose 6-phosphate-dependent and mannose 6-phosphate-independent lysosomal-targeting mechanisms have been described [10]. So, how might binding to VpslOp result in sorting of carboxypeptidase Y to the vacuole? Emr and colleagues suggest, by analogy with the mannose 6-phosphate receptor in mammalian cells, that VpslOp-p2CPY complexes are transported first to endosomes, where p2CPY is released and transported to the vacuole. VpslOp would then recycle back to the trans-Golgi compartment for binding further p2CPY molecules. The cytoplasmic tail of VpslOp contains potential targeting motifs that could direct its retrieval to the Golgi apparatus [1]. The other VPS gene products implicated in carboxypeptidase Y sorting are postulated to regulate such receptor-ligand traffic, but how do they do this? A tantalizing clue to the mechanism by which vacuolar protein sorting is regulated comes from earlier work of Emr and colleagues [11], which identified a key signalling complex required for vacuolar sorting. This is a complex between the serine/threonine protein kinase Vpsl5p and the phosphatidylinositol 3-kinase (PI 3-kinase) Vps34p. Emr and colleagues now suggest [1] that the VpslOp-p2CPY complex aggregates and locally stimulates the protein kinase activity of Vpsl5p (Fig. 2). This then activates Vps34p by phosphorylation, and Vps34p in turn phosphorylates phosphatidylinositol in the sorting compartment membrane. This may have the effect of physically stimulating membrane curvature, and thus facilitating the formation of transport vesicles containing VpslOp-p2CPY complexes destined for the prevacuolar compartment. Presumably, some sort of coat-protein complex must also be recruited at this stage, in order to form the vesicle that buds from the transGolgi network and fuses with the endosome. The specific carboxypeptidase-Y-specific sorting role of Vps29p and Vps35p may be in promoting the interaction between the VpslOp-p2CPY and Vps34p-Vpsl5p complexes, or alternatively they may be required for the specific recycling of VpslOp from the prevacuolar compartment to the trans-Golgi network. If this model is correct, one can envisage how other receptor-ligand complexes may act in the same way on Vps34p-Vpsl5p and the associated transport machinery. The demonstration of receptor-ligand-stimulated vesicle formation could have consequences beyond vacuolar protein sorting. Such a mechanism could locally stimulate traffic between organelles throughout the secretory pathway, in proportion to the amount of
Fig. 2. A model of the carboxypeptidase-Y-sorting mechanism in yeast. The complex between the carboxypeptidase Y receptor (vpsl Op) and the incompletely processed, Golgi form of carboxypeptidase Y (p2CPY) could associate with Vps35p and/or Vps29p, and transmits a signal to the Vpsl 5p-Vps34p complex. This could activate the protein kinase Vpsl 5p, which phosphorylates and activates Vps34p. Vps34p is a PI 3-kinase, which when activated phosphorylates the local membrane phosphatidylinositol lipids. This increases the local membrane curvature, which stimulates vesicle production.
ligand that needs to be sorted. However, not all sorting decisions within the secretory pathway can be explained this way. For example, the vacuolar membrane proteins seem to have no sorting signals, but are nevertheless efficiently sorted away from secreted proteins. Furthermore, resident Golgi glycosyltransferases are sorted in a poorly understood, sequence-independent manner. Determining how these different mechanisms are integrated to maintain the complex organization of the secretory pathway will keep researchers busy for a few years to come.
References: 1. Marcusson EG, Horazdovsky BF, Cereghino JL, Gharakhanian E, Emr SD: The sorting receptor for yeast vacuolar carboxypeptidase Y isencoded by the VPS10 gene. Cell 1994, 77:579-586. 2. Pelham HRB, Munro S: Sorting of membrane proteins in the secretory pathway. Cell 1993, 75:603-605. 3. Bretscher MS, Munro S: Cholesterol and the Golgi apparatus. Science 1993, 261:1280-1281. 4. Kornfield S: Structure and function of the mannose 6-phosphate/insulin-like growth factor II receptors. Annu Rev Biochem 1992, 61:307-330.
1021
1022
Current Biology 1994, Vol 4 No 11 5.
Raymond CK, Roberts C, Moore KE, Howald I, Stevens TH: Biogenesis of the vacuole in Saccharomyces cerevisiae. Int Rev Cytol 1992, 139:59-120. 6. Seeger M, Payne G: A role for clathrin in the sorting of vacuolar proteins in the Golgi complex of yeast. EMBO J 1992, 11:2811-2818. 7. Raymond CK, Howald I, Stevenson I, Vater CA, Stevens TH: Morphological classification of the yeast vacuolar protein sorting mutants: evidence for a prevacuolar compartment in class E vps mutants. Mo/ Biol Cell 1992, 3:389-1402 8. Stack JH, Emr SD: Genetic and biochemical studies of protein sorting to the yeast vacuole. Curr Opin Cell Biol 1993, 5:641-646 9. Paravacini G, Horazdovsky BF, Emr SD: Alternative pathways for
the sorting of soluble vacuolar proteins in yeast: a vps35 null mutant missorts and secretes only a subset of vacuolar hydrolases. Mol Biol Cell 1992, 3:415-427 10. Glickman JN, Kornfield S: Mannose 6-phosphate-independent targeting of lysosomal enzymes in I-cell disease B lymphoblasts. J Cell Biol 1993, 123:99-108 11. Stack JH, Herman PK, Schu PV, Emr SD: A membrane-associated complex containing the Vps15 protein kinase and the Vps34 PI 3-kinase is essential for protein sorting to the yeast lysosome-like vacuole. EMBO J 1993, 12:2195-2204
Rowan E. Chapman, MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2NQ, UK.
THE AUGUST 1994 ISSUE (VOL. 6, NO. 4) OF CURRENT OPINION IN CELL BIOLOGY included the following reviews, edited by Ira Mellman, on Membranes and Sorting: Post-translational protein import and folding by Jorg HBhfeld and E Ulrich Hartl Protein translocation across the endoplasmic reticulum by Davis T.W. Ng and Peter Walter Retention and retrieval in the endoplasmic reticulum and the Golgi apparatus by Tommy Nilsson and Graham Warren Rab GTPases: master regulators of membrane trafficking by Suzanne R. Pfeffer ARF: a key regulatory switch in intracellular membrane traffic and organelle structure by Julie G. Donaldson and Richard D. Klausner Coat proteins in intracellular membrane transport by Thomas E. Kreis and Rainer Pepperkok The role of clathrin, adaptors and dynamin in endocytosis by Margaret S. Robinson Mechanisms of cell polarity: sorting and transport in epithelial cells by Karl Matter and Ira Mellman Mechanisms of vesicle docking and fusion: insights from the nervous system byJonathan Pevsner and Richard H. Scheller Formation of synaptic vesicles by Olaf Mundigl and Pietro De Camilli The same issue also included the following reviews on Membrane Permeability edited by Lily Yeh Jan and Yuh Nung Jan: Bacterial transporters by Peter C. Maloney Mammalian exchangers and co-transporters by Reinhart A.F Reithmeier Neurotransmitter transporters: new members of known families by Patrick Schloss, Andreas W Piischel and Heinrich Betz Molecular physiology of anion channels by Thomas J. Jentsch Molecular properties of a superfamily of plasma-membrane cation channels by William A. Catterall
VACUOLAR SORTING
Tracking down an elusive receptor The receptor that sorts carboxypeptidase Y for transport to the vacuole in yeast has recently been identified; how vacuolar sorting is regulated is far from understood, but an intriguing model has been proposed. Understanding the complex organization and operation of the eukaryotic secretary pathway is an exciting challenge for cell biologists. How does transport occur between membrane-bound compartments within cells? And given the continuous flux between these compartments, how do they retain their distinct composition, and thus their identity? We now have a good idea about the routes proteins take through the secretary pathway, and about the amino-acid 'signal' sequences required for residence in most of the compartments. However, little is known about the actual sorting mechanism by which proteins carrying the appropriate signal sequences are recognized and directed to the appropriate compartment. The recent identification of the sorting receptor for the vacuolar protein carboxypeptidase Y in yeast [1] suggests that this state of ignorance may soon be a thing of the past. In both yeast and mammalian cells, sorting decisions must be made at every step along the secretary pathway (Fig. 1). According to the widely accepted 'bulk flow' hypothesis, no specific information is required for proteins to progress along the pathway to secretion from the cell. There must, therefore, be positive signals that prevent proteins destined for residence in a compartment along the pathway, or one reached by a branch off the
main pathway, from moving forward with the bulk flow. The first compartment in the pathway is the endoplasmic reticulum (ER), where proteins that take the secretary pathway are synthesized and folded, and where their glycosylation begins. The proteins then move to the Golgi apparatus, a series of membranous subcompartments in which further processing reactions - glycosylation, lipid modification and proteolysis - occur. The Golgi apparatus is where the first sorting decision is made, in which ER-resident proteins are separated from the rest and directed - presumably along with ER-specific lipids - back to the ER. The retention of ER-resident proteins thus operates via a retrieval mechanism, rather than by inhibition of their forward transport (reviewed in [2]). Specific amino-acid sequences that are necessary and sufficient for proteins to be retrieved in this way have been identified in both soluble and integral membrane ER proteins (the signals being, in the singleletter code, H/KDEL and KKXX, respectively) [2]. The retention of Golgi-resident glycosyltransferases, on the other hand, does not appear to rely on specific aminoacid sequences, depending rather on something about the length and composition of their transmembrane domains that has not yet been precisely defined [3]; as
Fig. 1. The eukaryotic secretory pathway. Movement between the different organelles is widely thought to occur via carrier vesicles. The yeast vacuole and mammalian lysosome share many features and are though to represent functionally equivalent compartments; both contain most of the cells' hydrolytic enzymes, have an acidic interior and are derived from a branch of the secretory pathway. Complete arrows indicate experimentally established transport steps; broken arrows indicate predicted, but not yet established, steps.
© Current Biology 1994, Vol 4 No 11
1019
1020
Current Biology 1994, Vol 4 No 11 yet there is no evidence that these enzymes are retained within the Golgi apparatus in a similar manner to the ER-resident proteins (that is, by a recycling mechanism). After passage through, and modification within, subcompartments of the Golgi apparatus, proteins move to the trans-Golgi network, a specialized processing and sorting compartment. Proteins resident in this compartment appear (like ER-resident proteins) to have specific amino-acid sequences that direct their retrieval from a post-Golgi compartment [2]. For those proteins destined to move on, the pathway bifurcates; they are either sorted to the lysosomal/vacuolar system or continue on the route to the plasma membrane. In both yeast and mammalian cells, specific signal sequences have been identified that are sufficient to direct soluble hydrolases to the lysosome/vacuole (reviewed in [4,5]). The situation is slightly more complicated in the case of integral membrane proteins; in mammalian cells, integral membrane proteins lacking a specific signal sequence accumulate at the plasma membrane, whereas in yeast they end up in the vacuole. However this apparent difference in 'default' localization is not completely clear-cut, as in mutant yeast cells with defects of the clathrin heavy chain, the trans-Golgi protein Kex2p is mislocalized to the plasma membrane rather than to the vacuole [6]. Further work is needed to sort out exactly what is going on. Despite this, many aspects of the secretory pathway compartments and sorting signals seem to be remarkably well conserved throughout the eukaryote kingdom. Much less is known about the molecular mechanisms involved in recognizing these compartmental 'address labels'. Until recently, only two receptor molecules that sort proteins away from the bulk flow within the pathway had been identified: erd2, the recycling receptor that recognizes the H/KDEL amino-acid signal on soluble ERresident proteins [2], and the mannose-6-phosphate receptor, which recognizes a specific sugar-phosphate linkage attached to a subset of mammalian hydrolases and directs the recognized enzymes to the lysosome [5]. Now, a third receptor has been identified in yeast [1]; this is a molecule that specifically recognizes the yeast enzyme carboxypeptidase Y and directs it to the vacuole. The identification of the carboxypeptidase Y receptor comes ten years after the initial genetic screens for yeast mutants defective in vacuolar protein sorting (known as vps mutants). The screens were set up to look for mutants that secrete the incompletely processed, Golgi form of carboxypeptidase Y (p2CPY), and are thus defective in sorting proteins from the trans-Golgi to the vacuole. The vps mutations identified in several screens were grouped into 45 complementation groups [7]. Many of these were found to be allelic with genes identified in screens for mutants with altered vacuolar morphology, Ca2 +-resistance and sensitivity to osmotic stress. Further analysis showed that as well as their p2CPY-secretion defect, most
of the mutants are defective in sorting the soluble vacuolar proteins proteinase A and proteinase B, although vacuolar membrane proteins seemed to stay inside the mutant cells. These pleiotropic phenotypes, along with the sheer number of complementation groups, indicate that many proteins are required for correct targeting of soluble vacuolar proteins (reviewed in [8]). Despite this complexity, there was evidence suggesting that amongst the VPS genes there would be one encoding a specific receptor for carboxypeptidase Y. Firstly, the carboxypeptidase-Y-sorting mechanism was shown to be saturable - over-expression of carboxypeptidase Y in wild-type cells results in its secretion from the cell. This is a specific effect, as proteinase A secretion does not result from over-expression of carboxypeptidase A, nor .does over-expression of proteinase A cause carboxypeptidase Y secretion. Sorting of carboxypeptidase Y relies on a specific amino-acid sequence (QRPL) at its amino terminus; changing this to KRPL abolishes sorting to the vacuole. With this information, Scott Emr and colleagues set out to find the carboxypeptidase Y receptor gene amongst the mass of carboxypeptidase-Y-secreting vps mutants. They found three mutant yeast strains, vpslO, vps35 and vps29, that appeared specifically to missort carboxypeptidase Y and to have no other defects [9]. Three genes were thus implicated in carboxypeptidase Y sorting, and the first of these to be cloned was VPS35. Despite the specificity of the carboxypeptidase-Y-sorting defect in the vps35 mutant, it was hard to see how VPS35 could encode the carboxypeptidase Y receptor as the gene product product, Vps35p, is peripherally associated with the cytoplasmic face of Golgi membranes [9], whereas the precursor form of carboxypeptidase Y, p2CPY, is a lumenal protein. Cloning of VPSIO was a different story. The recent paper from Emr and colleagues [1] shows quite clearly that VpslOp has the appropriate structure, intracellular location and p2CPYbinding properties to be a specific carboxypeptidase-Ysorting receptor. The large, 1413-residue VpslOp is an integral membrane protein located in the trans-Golgi sorting compartment. Furthermore, it has a large lumenal domain that can be specifically cross-linked to p2CPY. This interaction is dependant on the carboxypeptidase Y sorting signal, QRPL, as a sorting-deficient form (with QRPL changed to KRPL) cannot be cross-linked to p2CPY under the same conditions. The QRPL signal is sufficient to direct the association of a reporter molecule with VpslOp. Taken together, these results provide compelling evidence that VpslOp is a carboxypeptidase Y receptor. It seems unlikely, however, that VpslOp is a receptor exclusively for carboxypeptidase Y, as it can be specifically cross-linked to two further proteins, which could be hitherto unidentified VpslOp ligands. VpslOp is probably one member of a family of hydrolase receptors that includes sorting-receptors for proteinases A and B. These are unlikely to be encoded by any of the VPS genes, as
DISPATCH
there are no vps mutants that specifically missort either proteinase whilst retaining carboxypeptidase Y, the predicted phenotype of a mutation in a proteinase A or proteinase B receptor. Additional screens will therefore be required to identify further vacuolar-protein-sorting receptors. The occurrence in yeast of more than one vacuolar-targeting signal, and thus more than one sorting receptor, is similar to the situation in mammalian cells, where both mannose 6-phosphate-dependent and mannose 6-phosphate-independent lysosomal-targeting mechanisms have been described [10]. So, how might binding to VpslOp result in sorting of carboxypeptidase Y to the vacuole? Emr and colleagues suggest, by analogy with the mannose 6-phosphate receptor in mammalian cells, that VpslOp-p2CPY complexes are transported first to endosomes, where p2CPY is released and transported to the vacuole. VpslOp would then recycle back to the trans-Golgi compartment for binding further p2CPY molecules. The cytoplasmic tail of VpslOp contains potential targeting motifs that could direct its retrieval to the Golgi apparatus [1]. The other VPS gene products implicated in carboxypeptidase Y sorting are postulated to regulate such receptor-ligand traffic, but how do they do this? A tantalizing clue to the mechanism by which vacuolar protein sorting is regulated comes from earlier work of Emr and colleagues [11], which identified a key signalling complex required for vacuolar sorting. This is a complex between the serine/threonine protein kinase Vpsl5p and the phosphatidylinositol 3-kinase (PI 3-kinase) Vps34p. Emr and colleagues now suggest [1] that the VpslOp-p2CPY complex aggregates and locally stimulates the protein kinase activity of Vpsl5p (Fig. 2). This then activates Vps34p by phosphorylation, and Vps34p in turn phosphorylates phosphatidylinositol in the sorting compartment membrane. This may have the effect of physically stimulating membrane curvature, and thus facilitating the formation of transport vesicles containing VpslOp-p2CPY complexes destined for the prevacuolar compartment. Presumably, some sort of coat-protein complex must also be recruited at this stage, in order to form the vesicle that buds from the transGolgi network and fuses with the endosome. The specific carboxypeptidase-Y-specific sorting role of Vps29p and Vps35p may be in promoting the interaction between the VpslOp-p2CPY and Vps34p-Vpsl5p complexes, or alternatively they may be required for the specific recycling of VpslOp from the prevacuolar compartment to the trans-Golgi network. If this model is correct, one can envisage how other receptor-ligand complexes may act in the same way on Vps34p-Vpsl5p and the associated transport machinery. The demonstration of receptor-ligand-stimulated vesicle formation could have consequences beyond vacuolar protein sorting. Such a mechanism could locally stimulate traffic between organelles throughout the secretory pathway, in proportion to the amount of
Fig. 2. A model of the carboxypeptidase-Y-sorting mechanism in yeast. The complex between the carboxypeptidase Y receptor (vpsl Op) and the incompletely processed, Golgi form of carboxypeptidase Y (p2CPY) could associate with Vps35p and/or Vps29p, and transmits a signal to the Vpsl 5p-Vps34p complex. This could activate the protein kinase Vpsl 5p, which phosphorylates and activates Vps34p. Vps34p is a PI 3-kinase, which when activated phosphorylates the local membrane phosphatidylinositol lipids. This increases the local membrane curvature, which stimulates vesicle production.
ligand that needs to be sorted. However, not all sorting decisions within the secretory pathway can be explained this way. For example, the vacuolar membrane proteins seem to have no sorting signals, but are nevertheless efficiently sorted away from secreted proteins. Furthermore, resident Golgi glycosyltransferases are sorted in a poorly understood, sequence-independent manner. Determining how these different mechanisms are integrated to maintain the complex organization of the secretory pathway will keep researchers busy for a few years to come.
References: 1. Marcusson EG, Horazdovsky BF, Cereghino JL, Gharakhanian E, Emr SD: The sorting receptor for yeast vacuolar carboxypeptidase Y isencoded by the VPS10 gene. Cell 1994, 77:579-586. 2. Pelham HRB, Munro S: Sorting of membrane proteins in the secretory pathway. Cell 1993, 75:603-605. 3. Bretscher MS, Munro S: Cholesterol and the Golgi apparatus. Science 1993, 261:1280-1281. 4. Kornfield S: Structure and function of the mannose 6-phosphate/insulin-like growth factor II receptors. Annu Rev Biochem 1992, 61:307-330.
1021
1022
Current Biology 1994, Vol 4 No 11 5.
Raymond CK, Roberts C, Moore KE, Howald I, Stevens TH: Biogenesis of the vacuole in Saccharomyces cerevisiae. Int Rev Cytol 1992, 139:59-120. 6. Seeger M, Payne G: A role for clathrin in the sorting of vacuolar proteins in the Golgi complex of yeast. EMBO J 1992, 11:2811-2818. 7. Raymond CK, Howald I, Stevenson I, Vater CA, Stevens TH: Morphological classification of the yeast vacuolar protein sorting mutants: evidence for a prevacuolar compartment in class E vps mutants. Mo/ Biol Cell 1992, 3:389-1402 8. Stack JH, Emr SD: Genetic and biochemical studies of protein sorting to the yeast vacuole. Curr Opin Cell Biol 1993, 5:641-646 9. Paravacini G, Horazdovsky BF, Emr SD: Alternative pathways for
the sorting of soluble vacuolar proteins in yeast: a vps35 null mutant missorts and secretes only a subset of vacuolar hydrolases. Mol Biol Cell 1992, 3:415-427 10. Glickman JN, Kornfield S: Mannose 6-phosphate-independent targeting of lysosomal enzymes in I-cell disease B lymphoblasts. J Cell Biol 1993, 123:99-108 11. Stack JH, Herman PK, Schu PV, Emr SD: A membrane-associated complex containing the Vps15 protein kinase and the Vps34 PI 3-kinase is essential for protein sorting to the yeast lysosome-like vacuole. EMBO J 1993, 12:2195-2204
Rowan E. Chapman, MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2NQ, UK.
THE AUGUST 1994 ISSUE (VOL. 6, NO. 4) OF CURRENT OPINION IN CELL BIOLOGY included the following reviews, edited by Ira Mellman, on Membranes and Sorting: Post-translational protein import and folding by Jorg HBhfeld and E Ulrich Hartl Protein translocation across the endoplasmic reticulum by Davis T.W. Ng and Peter Walter Retention and retrieval in the endoplasmic reticulum and the Golgi apparatus by Tommy Nilsson and Graham Warren Rab GTPases: master regulators of membrane trafficking by Suzanne R. Pfeffer ARF: a key regulatory switch in intracellular membrane traffic and organelle structure by Julie G. Donaldson and Richard D. Klausner Coat proteins in intracellular membrane transport by Thomas E. Kreis and Rainer Pepperkok The role of clathrin, adaptors and dynamin in endocytosis by Margaret S. Robinson Mechanisms of cell polarity: sorting and transport in epithelial cells by Karl Matter and Ira Mellman Mechanisms of vesicle docking and fusion: insights from the nervous system byJonathan Pevsner and Richard H. Scheller Formation of synaptic vesicles by Olaf Mundigl and Pietro De Camilli The same issue also included the following reviews on Membrane Permeability edited by Lily Yeh Jan and Yuh Nung Jan: Bacterial transporters by Peter C. Maloney Mammalian exchangers and co-transporters by Reinhart A.F Reithmeier Neurotransmitter transporters: new members of known families by Patrick Schloss, Andreas W Piischel and Heinrich Betz Molecular physiology of anion channels by Thomas J. Jentsch Molecular properties of a superfamily of plasma-membrane cation channels by William A. Catterall