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Do GTPases direct membrane traffic in secretion?
Cell, Vol. 53, 669-671, J’une 3, 1988, Copyright
0 1988 by Cell Press
Do GTPases Direct Membrane Traffic in Secretion? Henry R. Bourne Departments of Pharmacology, Medicine, and the Cardiovascular Research Institute University of California San Francisco, California 94143-0450
Minireview
The Data
ylation of invertase, and decrease its secretion. Immunofluorescence studies (Segev et al.) show that antibodies directed against Yptlp stain Golgi structures in both yeast and mammalian cells. Taken together, these studies indicate that Yptlp resides in the Golgi and suggest that it plays a role in transpor? of proteins through that organelle. Mutations in a different yeast gene, SEC+ block a later step in secretion that occurs between the Golgi apparatus and the plasma membrane; the gene product, Sec4p, is a 23.5 kd GTP-binding protein whose amino acid sequence is 48% identical with that of Yptlp. Using antibodies, Goud et al. (this issue) find that 85% of Sec4p is tightly attached to the cytoplasmic faces of either post-Golgi secretory vesicles or the plasma memlbrane; the rest is soluble in the cytoplasm. Mutations in other set genes that block secretion at sites distal to the Golgi apparatus cause Sec4p to associate predominantly with the secretory vesicles that accumulate as a result of the block; set mutations that block at earlier stages (e.g., in the Golgi) do not affect the relative distribution of Sec4p between secretory vesicles and the plasma membrane. These observations indicate that the role of Sec4p in the secretory pathway is distal to that of Yptlp. Although these experiments localize structurally defined GTP-binding proteins in compartments of the secretory pathway, they do not identify specific biochemical functions for Yptlp and Sec4p. The fourth set of experiments (Melancon et al., opcit.), conducted in a mammalian system, does not identify a specific GTP-binding protein, but does reveal striking effects of a GTP analog on transport of vesicles within the Golgi apparatus. The cell-free experimental system assesses transport via vesicles that originate in “donor” Golgi, derived from cells which express the vesicular stomatitis virus glycoprotein (VSV-G) but lack N-acetylgiucosaminyltransferase I (GlcNAc). The transport vesicles attach to ‘acceptor” Golgi, derived from a wild-type cell with normal GlcNAc but lacking VSV-G. Subsequent fusion of the vescles with acceptor membranes makes VSV-G accessible for GlcNAccatalyzed incorporation of radiolabel from UDP-[3H]Nacetylglucosamine; the incorporated radiolabel serves as the measure of vesicle transport between1 cisternae of the Golgi stack. Melancon et al. find that this transport is inhibited by a hydrolysis-resistant GTP analog, GTP$. Transport is also inhibited by an aluminum fluoride complex that, like GTPyS, is known to activate the signaltransducing G proteins (Casey and Gilman, JBG 263, 2577-2580, 1988).
Three recent reports in Ce// use the behavior of mutants to implicate GTP-binding proteins as regulatory elements in the secretory pathway of the yeast Saccharomyces cerevisiae. The yeast YPT7 gene encodes a 23 kd protein, Yptlp, that binds and hydrolyzes GTP Segev et al. and Schmitt et al. (op. cit.) report that conditional lethal mutations of the YPT7 gene cause membranes and vesicles to accumulate within the yeast cell, prevent complete glycos-
The authors of all these papers interpret: their results in terms of G proteins and signal transduction. Goud et al. suggest that Sec4p may function as a G protein on the vesicle surface to transduce an intracellular signal needed to regulate transport between the Golgi apparatus and the plasma membrane. Melancon et al. suiggest that “the putative Golgi ‘G ’ protein” may be part of a superimposed
The growing superfamily of GTP-binding proteins has recently expanded to include proteins involved in secretion. How are these proteins related to known proteins that bind GTP? One model is provided by the ubiquitous family of G proteins that transduce hormonal and sensory signals across the plasma membrane. This would extend the distribution and function of G proteins from the surface of the cell to the interior. The GTP-binding proteins would mediate the effects of (unknown) regulatory stimuli on the rate of membrane transport at different steps of the secretory pathway (Melancon et al., Cell 51, 1053-1062,1987; Segev et al., Cell 52, 915924, 1988; Schmitt et al., Cell 53, 635-647, 1988; Goud et al., Ceil, this issue). Another model takes other GTP-binding proteins as a paradigm, and argues that the secretory GTP-binding proteins mediate vectorial transport of individual vesicles. In the bustling membrane traffic of the cell, these proteins could specify the direction traveled by each secretory (or other) vesicle between appropriate cellular compartments. I discuss this model in more detail below. First a necessary distinction: The term “G proteins” refers to the family of heterotrimeric proteins that transduce hormonal and sensory signals across the plasma membrane. The G proteins make up only one of several different families of GTP-binding proteins; other such families include the tubulins and the elongation and initiation factors of protein synthesis. The taxonomic distinction emphasizes a crucial point: Functionally, the signal-transducing G proteins represent only dne of several applications of the more general function of GTP binding and hydrolysis, which is to control switching between two different protein conformations. The switches are generally turned on in the GTP-bound form and turned off by hydrolysis (but not by dissociation) of GTP In the G proteins, the switch serves to propagate and amplify regulatory signals. Rather than regulate the rate or intensity of a reaction, however, the switch can serve instead to guarantee that a reaction proceeds in a single direction-as is the case, for example, in protein synthesis. Similarly, as proposed here, GTP/GDP-induced conformational switches may mediate vectorial transport of secretory vesicles.
The G Protein
Analogy
Cell 670
Donor Membrane
Chain Elongation
Acceptor
Membrane
B
regulatory mechanism that turns off constitutive transport, perhaps during mitosis, when vesicular transport is blocked. Lacking a pressing need for signal transduction in control of Golgi transport, however, these investigators cautiously aver that the putative GTP-binding protein could be part of the constitutive transport machinery. Segev et al. suggest that Yptlp “must transduce without external stimulus:’ because it is not located on the plasma membrane. They propose that Yptlp and its mammalian counterparts act as “labels” that signal the origin, destination, or contents of individual secretory vesicles; the processes responsible for affixing and reading the labels are not specified. A Different
Analogy
Indeed, as suggested by Segev et al., the most pressing question raised by developing knowledge of the secretory pathway concerns the mysterious mechanisms by which proteins destined for secretion are vectorially transported at every step in the process-from endoplasmic reticulum to Golgi, from one Golgi stack to another, from Golgi to secretory vesicles, and from secretory vesicles to the plasma membrane. Rather than the G proteins, the GTPbinding proteins of protein synthesis may provide the most relevant analogy for vectorial transport of secretory vesicles. Part A of the accompanying figure depicts the function of one of these proteins, elongation factor (EF) Tu. The GTP-bound form of EFTu binds aminoacyl-tRNA (aatRNA) and carries it to the A site of the ribosome-mRNA complex (labeled Ribo); if the aa-tRNA correctly matches the mRNA codon in the A site, EFTu hydrolyzes its bound GTP and dissociates from the ribosome, leaving the aatRNA attached and ready for the next step in elongation of the polypeptide chain (transfer to the ribosome’s P site). The cycle can begin again after another protein, EF-Ts, catalyzes exchange of GTP for the GDP bound to EF-Tu. Here the function of the GTP-dependent conformational switch is not to amplify a signal; instead, the switch promotes the energy-dependent, unidirectional transport of
one molecule to a binding site on another. Hydrolysis of the bound GTP performs two functions: it releases EFTu to participate in another cycle and shifts the binding equilibrium toward attachment of the aa-tRNA to the ribosome. By analogy to EF-Tu, a transport GTPase (X in part 6 of the figure) could mediate vectorial transport of vesicles between different membrane compartments. According to this hypothesis, Xorp recognizes a protein component(Y) of vesicles budding from the donor membrane. The XomY ternary complex on the vesicle’s surface specifies its attachment (via Y) to a docking protein (Z) on the appropriate acceptor membrane, whereupon hydrolysis of GTP releases Xepe, leaving the vesicle-YZ complex in a position to initiate the process of membrane fusion. XGDP is then recycled by conversion back to Xe~p, a reaction catalyzed by a guanine nucleotide exchange protein (EP) located on the donor membrane. In this scheme the Y component, not the GTP-binding protein, serves as the vesicle label postulated by Segev et al. The GTP-binding protein-X, Yptl, Sec4p, etc.ensures that the labeled package is deposited at the appropriate address (Z). For example, the GTP-bound form of Sec4p (= Xo~p) would recognize Y on post-Golgi secretory vesicles and promote their specific attachment to Z, located on the plasma membrane; following GTP hydrolysis, Sec4p would return through the cytoplasm to target another secretory vesicle for transport to the plasma membrane, while the YZ complex would set the stage for fusion of vesicle and plasma membrane, resulting in exocytosis. This hypothesis readily explains why high concentrations of GTP block inhibition of vesicle transport by GTPys in the experiments of Melancon et al. This is precisely the expected result if hydrolysis of GTP allows recycling of X, Y, and Z components and continuing transport between Golgi stacks, whereas the hydrolysis-resistant GTP analog blocks transport by causing accumulation of Y and Z bound irreversibly to Xorpys. The hypothesis depicted in
MinIreview 671
part B of the figure is consistent with all the data thus far. indeed, the EF-Iu model directly predicts several otherwise puzzling observations about the inhibitory effect of GTPyS, including the following: (i) it requires simultaneous incubation with both membranes and cytosol; (ii) it is irreversible; (iii) it is exerted at the acceptor rather than the donor membrane; and (iv) it is accompanied by an increase (seen in electron micrographs) in the number of unfused vesicles and buds associated with the Golgi apparatus. In the signal transduction model, the “inhibitory factor” in cytosol is proposed to be separate from the constitutive transport machinery, on the basis of two observations. First, lower concentrations of cytosol are required to support maximal transport than are required for maximal inhibition by GTP+; and, second, incubation of cytosol with GTPvS and membranes partially depletes the cytosol’s ability to support GTPyS-dependent inhibition of transport in subsequent inhibitions with fresh membranes. Both these observations are also consistent with the model depicted in the figure-i.e., that the inhibitory factor is a component of the constitutive transport machinery-if we assume that endogenous GTP (present in the incubations) competes with GTPyS for binding to the transport GTPase. In this case, GTP allows low concentrations of X (i.e., of cytosol) to catalyze a brisk rate of vesicle transport because limiting components (e.g., Z) can recycle back into the transport system. In contrast, inhibition of transport by GTP$ requires high concentrations of X (i.e., of cytosol), because the inhibition results from stoichiometric sequestration of a limiting component, presumably Z, in the XG~~+YZ complex; this explains why GTPrS inhibits at the acceptor membrane and also why membranes can deplete cytosol of “inhibitory factor” (= X) in I) the presence of GTPyS. If the GTPase model proves valid for one specific example of vesicle transport, the same biochemical mechanism may turn out to mediate other steps in the secretory pathway and-perhaps-other kinds of membrane traffic as well (e.g., in endocytosis). If so, the requirement for multiple transport GTPases could begin to account for the growing number of small (20-25 kd) GTP-binding proteins found in mammalian cells; not counting the three c-ras gene products, distinct cDNAs have been reported for at least eight such proteins, including four mammalian relatives of yeast Yptlp (Touchot et al., PNAS 84, 8210-8214,
1987; Haubruck et al., EMBO J. 6, 4049-4053, 1987). In addition, several botulinum neurotoxins, which block both spontaneous and evoked secretion of meurotransmitters, catalyze GTP-sensitive ADP-ribosylatilon of proteins of similar size (22-26 kd) (Matsuoka et al., FEBS Lett. 276, 295-299, 1987; Rosener et al., ibid, 224, 38-42, 1987); these proteins also may turn out to be transport GTPases. As is often the case, however, there rnay be a fly in the ointment. Altt)ough in general agreement with Segev et al. on the secretory phenotype produced by yptl mutations, Schmitt et al. (op. cit.) report two additional findings. First, mutational inactivation of Yptlp causes the yeast cells to take up radioactive calcium at a greatly increased rate, probably indicating a decreased concentration of cytoplasmic Ca2+. Second, and more surprisingly, addition of 50 m M Ca2+ to the medium partially reverses the growth inhibition caused by the yptl mutation at the restrictive temperature. Schmitt et al. suggest that the primary function of Yptlp is to regulate cytoplasmic Ca*+ concentration, rather than vesicular transport through the Golgi. If so, the yptl secretion defect and the accumulation of intracellular membranes ought to be ameliorated in cells grown in 50 m M Ca2+ -a prediction not yet tested. An alternative interpretation turns the argument around: The decreased cytoplasmic Ca2+ (and the growth defect) may result from increased sequestration of the ion in the greatly increased number and volume of intracellular vesicles that accumulate in yptl mutant cells; in this case, addition of Caz+ to the medium would not reverse the secretory defect of yptl mutants. The latter view, of course, is consistent with a primary role of Yptlp as a GTPase involved in vesicle transport. In summary, it should be emphasized that only further experiments will decide whether G protein-mediated signaling or vectorial transport by GTPases is the correct model for the functions of Yptlp, Sec4p, or the protein that inhibits Golgi transport in the presence of GTPyS and aluminum fluoride. It is quite possible that neither model will fit the case. Indeed, the discovery of a new GTP-binding protein need not force us to choose a model from the limited set of established precedents. Instead, it may prove wiser to focus first on the underlying principle-i.e., that GTP binding and hydrolysis are used to produce two different protein conformations-realizing that evolution may have found even more creative ways of putting these conformations to use.
0 1988 by Cell Press
Do GTPases Direct Membrane Traffic in Secretion? Henry R. Bourne Departments of Pharmacology, Medicine, and the Cardiovascular Research Institute University of California San Francisco, California 94143-0450
Minireview
The Data
ylation of invertase, and decrease its secretion. Immunofluorescence studies (Segev et al.) show that antibodies directed against Yptlp stain Golgi structures in both yeast and mammalian cells. Taken together, these studies indicate that Yptlp resides in the Golgi and suggest that it plays a role in transpor? of proteins through that organelle. Mutations in a different yeast gene, SEC+ block a later step in secretion that occurs between the Golgi apparatus and the plasma membrane; the gene product, Sec4p, is a 23.5 kd GTP-binding protein whose amino acid sequence is 48% identical with that of Yptlp. Using antibodies, Goud et al. (this issue) find that 85% of Sec4p is tightly attached to the cytoplasmic faces of either post-Golgi secretory vesicles or the plasma memlbrane; the rest is soluble in the cytoplasm. Mutations in other set genes that block secretion at sites distal to the Golgi apparatus cause Sec4p to associate predominantly with the secretory vesicles that accumulate as a result of the block; set mutations that block at earlier stages (e.g., in the Golgi) do not affect the relative distribution of Sec4p between secretory vesicles and the plasma membrane. These observations indicate that the role of Sec4p in the secretory pathway is distal to that of Yptlp. Although these experiments localize structurally defined GTP-binding proteins in compartments of the secretory pathway, they do not identify specific biochemical functions for Yptlp and Sec4p. The fourth set of experiments (Melancon et al., opcit.), conducted in a mammalian system, does not identify a specific GTP-binding protein, but does reveal striking effects of a GTP analog on transport of vesicles within the Golgi apparatus. The cell-free experimental system assesses transport via vesicles that originate in “donor” Golgi, derived from cells which express the vesicular stomatitis virus glycoprotein (VSV-G) but lack N-acetylgiucosaminyltransferase I (GlcNAc). The transport vesicles attach to ‘acceptor” Golgi, derived from a wild-type cell with normal GlcNAc but lacking VSV-G. Subsequent fusion of the vescles with acceptor membranes makes VSV-G accessible for GlcNAccatalyzed incorporation of radiolabel from UDP-[3H]Nacetylglucosamine; the incorporated radiolabel serves as the measure of vesicle transport between1 cisternae of the Golgi stack. Melancon et al. find that this transport is inhibited by a hydrolysis-resistant GTP analog, GTP$. Transport is also inhibited by an aluminum fluoride complex that, like GTPyS, is known to activate the signaltransducing G proteins (Casey and Gilman, JBG 263, 2577-2580, 1988).
Three recent reports in Ce// use the behavior of mutants to implicate GTP-binding proteins as regulatory elements in the secretory pathway of the yeast Saccharomyces cerevisiae. The yeast YPT7 gene encodes a 23 kd protein, Yptlp, that binds and hydrolyzes GTP Segev et al. and Schmitt et al. (op. cit.) report that conditional lethal mutations of the YPT7 gene cause membranes and vesicles to accumulate within the yeast cell, prevent complete glycos-
The authors of all these papers interpret: their results in terms of G proteins and signal transduction. Goud et al. suggest that Sec4p may function as a G protein on the vesicle surface to transduce an intracellular signal needed to regulate transport between the Golgi apparatus and the plasma membrane. Melancon et al. suiggest that “the putative Golgi ‘G ’ protein” may be part of a superimposed
The growing superfamily of GTP-binding proteins has recently expanded to include proteins involved in secretion. How are these proteins related to known proteins that bind GTP? One model is provided by the ubiquitous family of G proteins that transduce hormonal and sensory signals across the plasma membrane. This would extend the distribution and function of G proteins from the surface of the cell to the interior. The GTP-binding proteins would mediate the effects of (unknown) regulatory stimuli on the rate of membrane transport at different steps of the secretory pathway (Melancon et al., Cell 51, 1053-1062,1987; Segev et al., Cell 52, 915924, 1988; Schmitt et al., Cell 53, 635-647, 1988; Goud et al., Ceil, this issue). Another model takes other GTP-binding proteins as a paradigm, and argues that the secretory GTP-binding proteins mediate vectorial transport of individual vesicles. In the bustling membrane traffic of the cell, these proteins could specify the direction traveled by each secretory (or other) vesicle between appropriate cellular compartments. I discuss this model in more detail below. First a necessary distinction: The term “G proteins” refers to the family of heterotrimeric proteins that transduce hormonal and sensory signals across the plasma membrane. The G proteins make up only one of several different families of GTP-binding proteins; other such families include the tubulins and the elongation and initiation factors of protein synthesis. The taxonomic distinction emphasizes a crucial point: Functionally, the signal-transducing G proteins represent only dne of several applications of the more general function of GTP binding and hydrolysis, which is to control switching between two different protein conformations. The switches are generally turned on in the GTP-bound form and turned off by hydrolysis (but not by dissociation) of GTP In the G proteins, the switch serves to propagate and amplify regulatory signals. Rather than regulate the rate or intensity of a reaction, however, the switch can serve instead to guarantee that a reaction proceeds in a single direction-as is the case, for example, in protein synthesis. Similarly, as proposed here, GTP/GDP-induced conformational switches may mediate vectorial transport of secretory vesicles.
The G Protein
Analogy
Cell 670
Donor Membrane
Chain Elongation
Acceptor
Membrane
B
regulatory mechanism that turns off constitutive transport, perhaps during mitosis, when vesicular transport is blocked. Lacking a pressing need for signal transduction in control of Golgi transport, however, these investigators cautiously aver that the putative GTP-binding protein could be part of the constitutive transport machinery. Segev et al. suggest that Yptlp “must transduce without external stimulus:’ because it is not located on the plasma membrane. They propose that Yptlp and its mammalian counterparts act as “labels” that signal the origin, destination, or contents of individual secretory vesicles; the processes responsible for affixing and reading the labels are not specified. A Different
Analogy
Indeed, as suggested by Segev et al., the most pressing question raised by developing knowledge of the secretory pathway concerns the mysterious mechanisms by which proteins destined for secretion are vectorially transported at every step in the process-from endoplasmic reticulum to Golgi, from one Golgi stack to another, from Golgi to secretory vesicles, and from secretory vesicles to the plasma membrane. Rather than the G proteins, the GTPbinding proteins of protein synthesis may provide the most relevant analogy for vectorial transport of secretory vesicles. Part A of the accompanying figure depicts the function of one of these proteins, elongation factor (EF) Tu. The GTP-bound form of EFTu binds aminoacyl-tRNA (aatRNA) and carries it to the A site of the ribosome-mRNA complex (labeled Ribo); if the aa-tRNA correctly matches the mRNA codon in the A site, EFTu hydrolyzes its bound GTP and dissociates from the ribosome, leaving the aatRNA attached and ready for the next step in elongation of the polypeptide chain (transfer to the ribosome’s P site). The cycle can begin again after another protein, EF-Ts, catalyzes exchange of GTP for the GDP bound to EF-Tu. Here the function of the GTP-dependent conformational switch is not to amplify a signal; instead, the switch promotes the energy-dependent, unidirectional transport of
one molecule to a binding site on another. Hydrolysis of the bound GTP performs two functions: it releases EFTu to participate in another cycle and shifts the binding equilibrium toward attachment of the aa-tRNA to the ribosome. By analogy to EF-Tu, a transport GTPase (X in part 6 of the figure) could mediate vectorial transport of vesicles between different membrane compartments. According to this hypothesis, Xorp recognizes a protein component(Y) of vesicles budding from the donor membrane. The XomY ternary complex on the vesicle’s surface specifies its attachment (via Y) to a docking protein (Z) on the appropriate acceptor membrane, whereupon hydrolysis of GTP releases Xepe, leaving the vesicle-YZ complex in a position to initiate the process of membrane fusion. XGDP is then recycled by conversion back to Xe~p, a reaction catalyzed by a guanine nucleotide exchange protein (EP) located on the donor membrane. In this scheme the Y component, not the GTP-binding protein, serves as the vesicle label postulated by Segev et al. The GTP-binding protein-X, Yptl, Sec4p, etc.ensures that the labeled package is deposited at the appropriate address (Z). For example, the GTP-bound form of Sec4p (= Xo~p) would recognize Y on post-Golgi secretory vesicles and promote their specific attachment to Z, located on the plasma membrane; following GTP hydrolysis, Sec4p would return through the cytoplasm to target another secretory vesicle for transport to the plasma membrane, while the YZ complex would set the stage for fusion of vesicle and plasma membrane, resulting in exocytosis. This hypothesis readily explains why high concentrations of GTP block inhibition of vesicle transport by GTPys in the experiments of Melancon et al. This is precisely the expected result if hydrolysis of GTP allows recycling of X, Y, and Z components and continuing transport between Golgi stacks, whereas the hydrolysis-resistant GTP analog blocks transport by causing accumulation of Y and Z bound irreversibly to Xorpys. The hypothesis depicted in
MinIreview 671
part B of the figure is consistent with all the data thus far. indeed, the EF-Iu model directly predicts several otherwise puzzling observations about the inhibitory effect of GTPyS, including the following: (i) it requires simultaneous incubation with both membranes and cytosol; (ii) it is irreversible; (iii) it is exerted at the acceptor rather than the donor membrane; and (iv) it is accompanied by an increase (seen in electron micrographs) in the number of unfused vesicles and buds associated with the Golgi apparatus. In the signal transduction model, the “inhibitory factor” in cytosol is proposed to be separate from the constitutive transport machinery, on the basis of two observations. First, lower concentrations of cytosol are required to support maximal transport than are required for maximal inhibition by GTP+; and, second, incubation of cytosol with GTPvS and membranes partially depletes the cytosol’s ability to support GTPyS-dependent inhibition of transport in subsequent inhibitions with fresh membranes. Both these observations are also consistent with the model depicted in the figure-i.e., that the inhibitory factor is a component of the constitutive transport machinery-if we assume that endogenous GTP (present in the incubations) competes with GTPyS for binding to the transport GTPase. In this case, GTP allows low concentrations of X (i.e., of cytosol) to catalyze a brisk rate of vesicle transport because limiting components (e.g., Z) can recycle back into the transport system. In contrast, inhibition of transport by GTP$ requires high concentrations of X (i.e., of cytosol), because the inhibition results from stoichiometric sequestration of a limiting component, presumably Z, in the XG~~+YZ complex; this explains why GTPrS inhibits at the acceptor membrane and also why membranes can deplete cytosol of “inhibitory factor” (= X) in I) the presence of GTPyS. If the GTPase model proves valid for one specific example of vesicle transport, the same biochemical mechanism may turn out to mediate other steps in the secretory pathway and-perhaps-other kinds of membrane traffic as well (e.g., in endocytosis). If so, the requirement for multiple transport GTPases could begin to account for the growing number of small (20-25 kd) GTP-binding proteins found in mammalian cells; not counting the three c-ras gene products, distinct cDNAs have been reported for at least eight such proteins, including four mammalian relatives of yeast Yptlp (Touchot et al., PNAS 84, 8210-8214,
1987; Haubruck et al., EMBO J. 6, 4049-4053, 1987). In addition, several botulinum neurotoxins, which block both spontaneous and evoked secretion of meurotransmitters, catalyze GTP-sensitive ADP-ribosylatilon of proteins of similar size (22-26 kd) (Matsuoka et al., FEBS Lett. 276, 295-299, 1987; Rosener et al., ibid, 224, 38-42, 1987); these proteins also may turn out to be transport GTPases. As is often the case, however, there rnay be a fly in the ointment. Altt)ough in general agreement with Segev et al. on the secretory phenotype produced by yptl mutations, Schmitt et al. (op. cit.) report two additional findings. First, mutational inactivation of Yptlp causes the yeast cells to take up radioactive calcium at a greatly increased rate, probably indicating a decreased concentration of cytoplasmic Ca2+. Second, and more surprisingly, addition of 50 m M Ca2+ to the medium partially reverses the growth inhibition caused by the yptl mutation at the restrictive temperature. Schmitt et al. suggest that the primary function of Yptlp is to regulate cytoplasmic Ca*+ concentration, rather than vesicular transport through the Golgi. If so, the yptl secretion defect and the accumulation of intracellular membranes ought to be ameliorated in cells grown in 50 m M Ca2+ -a prediction not yet tested. An alternative interpretation turns the argument around: The decreased cytoplasmic Ca2+ (and the growth defect) may result from increased sequestration of the ion in the greatly increased number and volume of intracellular vesicles that accumulate in yptl mutant cells; in this case, addition of Caz+ to the medium would not reverse the secretory defect of yptl mutants. The latter view, of course, is consistent with a primary role of Yptlp as a GTPase involved in vesicle transport. In summary, it should be emphasized that only further experiments will decide whether G protein-mediated signaling or vectorial transport by GTPases is the correct model for the functions of Yptlp, Sec4p, or the protein that inhibits Golgi transport in the presence of GTPyS and aluminum fluoride. It is quite possible that neither model will fit the case. Indeed, the discovery of a new GTP-binding protein need not force us to choose a model from the limited set of established precedents. Instead, it may prove wiser to focus first on the underlying principle-i.e., that GTP binding and hydrolysis are used to produce two different protein conformations-realizing that evolution may have found even more creative ways of putting these conformations to use.