TIBS
17
-
SEPTEMBER 1992
JOURNALCLUB One of the most fashionable drugs in recent membrane traffic research has been brefeldin A. This drug causes the 'disappearance' of the Golgi complex, and the return of Golgi markers to the endoplasmic reticulum (ER) (for review see Ref. 1). The loss of a well-defined, compact Golgi structure has been observed previously, for example during mitosis 2, but the mechanism for Golgi disassembly is still not at all clear. Three recent papers 3-5 may shed light on the mechanisms involved in Golgi stability and maintenance. In one 3, a process indistinguishable from that observed after brefeldin A treatment was described upon overexpression of a putative ER salvage receptor. In the other two papers 4'5, Golgi disappearance was observed in cells expressing temperature-sensitive secretion-defective mutations in the End4 complementation group.
Membrane traffic between the ER and Golgi The ER represents the entry site of the secretory pathway. Secretory and membrane proteins are transferred from the ER to the Golgi during forward/ anterograde membrane traffic, whereas resident ER proteins or transportincompetent proteins are retained 6. A recycling pathway from the Golgi to the ER to retrieve any proteins that have escaped ER retention appears to be exaggerated by the drug brefeldin A1. This type of transport is termed reverse or retrograde membrane traffic, since it operates against the bulk flow7 of membrane and secretory proteins out of the cell. In order to maintain the structure of organelles along the secretory pathway, tight controls of forward and reverse traffic must exist. Brefeldin A perturbs the control of membrane traffic in a most profound fashion, most • probably by increasing retrograde while blocking anterograde transport, thus causing the disappearance of the Golgi complexL
Golgi appearance and disappearance In the electron microscope the Golgi complex has been shown to consist of several distinct sets of stacked cisternae connected by tubules 8 while in im© 1992, Elsevier Science Publishers, (UK)
Now you see it, now you don't: the Golgi disappearing act munofluorescence microscopy it is observed as a compact juxtanuclear structure in the centrosome region 9. The 'disappearance' of the Golgi is defined here as the loss of a compact juxtanuclear structure when observed by indirect immunofluorescence microscopy. After Golgi disappearance, staining of Golgi markers may be punctate, diffuse or in a well-dispersed reticulum, which is indistinguishable from the ER. Previous studies of the disassembled Golgi complex during mitosis and during treatment with phosphatase inhibitors such as okadaic acid have led to the identification of Golgi remnants 2,~°that represent the end point of Golgi disassembly and are thought to be the template for Golgi reassembly. This disassembly leads to a block in transport to and through the Golgi complex. Golgi dispersal is also seen in the presence of microtubule-disrupting drugs 11. Here, intact Golgi stacks disperse from their normal position clustered around the centrosome. There is little evidence that such a dispersal impairs intracellular transport through the Golgi to any great extent, though there may be some effects on exit from the Golgi in these cells. The final type of Golgi disappearance is observed in cells treated with the fungal metabolite brefeldin A1. In this case, the Golgi complex appears to be disassembled by the formation of tubules, which emanate from Golgi cisternae and fuse with the ER, eventually leading to the complete redistribution of Golgi markers and enzymes to the ER, where they can act on resident ER proteins. This also leads to a block in the secretory pathway, probably at the level of export from the mixed ER-Golgi compartment.
Overexpression leads to Golgi disappearance Hsu e t al. 3 looked at the effects of overexpression of a human homologue of the ERD-2 ER salvage receptoP 2 and a closely related protein, which they named ELP-1. Using transient expression systems they looked for cells expressing varying levels of ELP-1 or ERD-2. In cells that expressed high levels of ELP-1 or ERD-2 an ER-type staining pattern was observed in immunofluorescence, whereas in the cells expressing lower levels of ELP-1 or ERD-2 a compact Golgi-like distribution was observed. Double-labelling experiments with a general Golgi marker, lentil lectin, showed that when ELP-I was found in an ER-like pattern, the lectin was found to stain in a similar fashion, rather than having its usual compact Golgi distribution. This redistribution was reminiscent of the changes induced by the drug brefeldin A. One of the first known effects of brefeldin A is the redistribution of [3-COP, a non-clathrin-coated vesicle coat protein ~3, to the cytosol. When the distribution of [~-COP was examined in cells overexpressing ELP-1 it was found to have become diffuse. Further effects of the overexpression of ELP-1 were a defect in secretion of cotransfected lysozyme and the induction of Golgitype processing of a cotransfected ER glycoprotein. All of these findings are similar to the known effects of brefeldin A. In order to explain why the overexpression of a putative salvage receptor induces a brefeldin A-like phenotype the authors propose a model in which overexpression of ELP-1 leads to the enhancement of retrograde traffic from the Golgi to the ER due to localized uncoating of Golgi membranes which contain ELP-1. If a high concentration of the salvage receptor leads to the release of Golgi coat proteins and the uncoated vesicles and tubules produced can participate in retrograde, but ~not anterograde transport between the ER and Golgi complex, this would lead to an imbalance in traffic between the ER and the Golgi and finally to a steadystate redistribution of Golgi compo, nents to the ER (Fig. 1).
325
TIBS 17 - SEPTEMBER 1992
(a)
¢._ ~ b q
-
~
(c)
(b) ~oGolgi
~O
.) O Golgi
C
~ o ~--
o Golgi ~-~ remnants?
ELP-I@?
I i•End4@?
Brefeldin A@ ..... End4EQ Brefeldin A@
Brefeldin A@
ER
)C
~
~
~
ER-GoIgi
~ Figure 1 Forward and reverse traffic between the ER and Golgi. (a) At steady state, in the absence of drug or mutant induction, forward and reverse traffic between the ER and Golgi are balanced, which maintains the well-defined structure of the Golgi complex. Separate ER and Golgi coated vesicles probably mediate forward traffic, while return traffic is thought to be via uncoated vesicles. Functional End4 protein may be involved in mediating transport from the ER 'to the Golgi. (b) A short time after the addition of brefeldin A or after mutant induction, retrograde transport via uncoated elements from the Golgi to the ER is enhanced while forward traffic is blocked, possibly due to inhibition of coat protein binding. Overexpression of ELP-1 might assist in the increase in retrograde traffic, while lack of functional End4 protein might block export from the ER. Brefeldin A induces the formation of retrograde tubules which are thought to mediate reverse traffic from the Golgi to the ER and at the same time seems to inhibit forward traffic. All of these effects might be mediated by an inhibition of coat protein binding to ER or Golgi membranes. (¢) After longer times in the presence of drug or after mutant induction a new steady state is reached. The Golgi has disappeared to form a miXed Golgi-ER organelle, possibly leaving Golgi remnants that are incapable of participating in membrane traffic. Lack of functional End4 protein or the presence of brefeldin A continues to block export from the ER. This condition can be reversed upon removal of brefeldin A or return to the permissive temperature.
Mutations leading to Golgi disappearance In the other two papers in which Golgi disappearance was observed the effects of temperature-sensitive secretion-defective mutations were examined. Zuber et al2 looked at a temperature-sensitive mutant cell line, DS28-6, using immunofluorescence and electron microscopy. They observed the redistribution of Golgi markers from a compact juxtanuclear cisternal structure to a more diffuse distribution. They also provided some evidence that at the non-permissive temperature some Golgi markers were found in the ER. Looking at another mutant cell line, V.24.1, in the same complementation group (End4), Kao and Draper 5 also found that typical Golgi morphology was lost at high temperatures. However, Golgi enzymes did not appear to become functional components of the ER and immunofluorescence did not reveal Golgi markers in an ER staining pattern. They also examined the site of the block in secretion which appeared to be at the exit site from the intermediate compartment 14,15 between the ER and Golgi. Golgi disassembly appeared to be assisted by an intact microtubute system (a phenomenon also observed during brefeldin A action) and by continuing forward traffic through the Golgi.
326
Viewing the structure of the Golgi and the ER as the products of a steadystate balance between forward and reverse traffic between the two compartments the findings in these two papers may not be incompatible. If the intermediate compartment is morphologically similar to the ER, but its contents are biochemically distinct from those of the ER, EM morphology could localize Golgi components to the ER while Golgi enzymes could n o t act on bona fide ER markers.
Models of Golgi stability The papers discussed above raise some important questions about'-the nature of controls governing membrane traffic between the ER and the Golgi complex. How is the structure of the Golgi complex maintained in most ceils in the absence of drug or mutant perturbations? How can overexpression of one protein and inactivation of another lead to very similar phenotypes? How similar are these two phenotypes? What might be the function and distribution of the proteins corresponding to the End4 complementation group, and does it interact with the salvage receptors or Golgi coat proteins? In the End4 mutants at the non-permissive temperature, what is the effect on the salvage receptors and on the coat proteins and
how rapid is the onset of any affects found? Figure 1 outlines some possible sites of action and control of the proteins involved in maintaining the balance between forward and reverse traffic between the ER and Golgi. Blocking exit from the ER or increasing return from the Golgi could both lead to the establishment of a combined ER-Golgi compartment. The model can be increased in complexity when we consider that there may need to be an intermediate compartment between the ER and Golgi. This would probably then be the site from which recycling would usually occur, and in the End4 mutant appears to be the site where secretory proteins are blocked. The level of interaction between the ER and intermediate compartment and between the Golgi and the intermediate compartment is currently under investigation in several laboratories, and their findings will probably refine our understanding of the End4 les'ion. Research into intracellular transport is very active at the moment and candidate proteins involved in membrane traffic are coming to light at an astonishing rate. Our picture of the processes involved in intracellular membrane traffic and in the maintenance of stable organelles within the context of
TIBS 17 - SEPTEMBER 1992 very extensive membrane flow through the pathway is becoming clearer. It will be important to continue studies using the combined approaches of molecular genetics, cell biology on intact cells and cell-free assays of intracellular transport to continue the rapid advances in our knowledge of these mechanisms. References 1 Klausner, R. D., Donaldson, J. G. and LippincottSchwartz, J. (1992) J. Cell Biol. 116, 1071-1080 2 Lucocq, J. M. and Warren, G. (1988) EMBO J.
6, 3239-3246 3 Hsu, V. W., Shah, N. and Klausner, R. D. (1992) Cell 69, 625-635 4 Zuber, C. et al. (1991) Proc. Natl Acad. Sci. USA 88, 9818-9822 5 Kao, C-Y. and Draper, R. K. (1992) J. Cell Biol. 117,701-715 6 Pfeffer, S. R. and Rothman, J. E. (1987) Annu. Rev. Biochem. 56, 829-852 7 HuRley, S. M. and Helenius, A. (1989) Annu. R~v. Cell Biol. 5, 277-307 8 Rambourg, A. and Clermont, Y. (1990) Eur. J. Cell Biol. 51, 189-200 9 Burke, B. et al. (1982) EMBO J. 1, 1621-1628 10 Lucocq, J., Warren, G. and Pryde, J. (1991) J. Cell Sci. 100, 753-759 11 Ho, W. C. et al. (1989) Eur. J. Cell Biol. 48,
250-263 12 Semenza, J. C., Hardwick, K. G., Dean, N. and Pelham, H. R. B. (1990) Cell 61, 1349-1357 13 HuRley, S. M. (1991) Trends Biochem. Sci. 16,
165-166 14 Schweizer, A. eta/. (1990) Eur. J. Cell Biol. 53,
185-196 15 Saraste, J. and Svensson, K. (1991) J. Cell Sci.
100, 415-430
STELLA M. HURTLEY Department of Biochemistry,Universityof Edinburgh Medical School, Hugh Robson Building, GeorgeSquare, Edinburgh,UK EH8 9XD.
'z
OBITUARY Shosaku Numa, Professor, Department of Medical Chemistry and Department of Molecular Genetics, Kyoto University Faculty of Medicine, Kyoto, Japan, passed away on February 15, 1992, about a month and a half before his formal retirement and after a nearly threeyear struggle with colon cancer and metastasis. Professor Numa was born on February 7, 1927, in Wakayama, Japan. In 1952, he received his MD from Kyoto University, and after having spent about 12 months in internship, he entered the First Department of Internal Medicine, Kyoto University Faculty of Medicine, for clinical training. However, he was not too happy about the quality of research in clinical medicine and in 1956 received a Fullbright Fellowship to work at the Department of Biological Chemistry, Harvard Medical School, Boston, where he carried out analytical work on serum lipoproteins under the guidance of Professor Oncley. In 1958, he moved to the Max-Planck-Instit0t fiir Zellchemie, Munich, as an Alexander yon Humboldt stipendiat and started to work on enzymology and metabolic regulation of lipids under the guidance of Professor F. Lynen. Three years later, he returned to Japan and was appointed an instructor in the Department of Medical Chemistry, Kyoto University Faculty of Medicine, and was soon promoted to associate professor. While continuing his own project on lipid metabolism, he also collaborated with me and made a large contribution towards the elucidation of the metabolic pathway of tryptophan in mam© 1992, Elsevier Science Publishers, (UK)
Shosaku Numa 1927-1992
mals and microorganisms. In 1963, he took a leave of absence to resume collaboration with Lynen, and in 1968, returned to Kyoto to become Professor in charge of the 2nd Department of Medical Chemistry. During the next ten years or so, he continued to work on lipid metabolism and, together with a large number of collaborators, published many important papers concerning the ,enzymes involved in fat metabolism, especially acetyl-CoA carboxylase, which included
their structures and regulatory mechanisms. In the early 1980s, his interest gradually shifted to the application of molecular biology techniques to the elucidation of the primary structure of neurotransmitter receptors and ionic channels. Professor Numa then cloned and sequenced the cDNA and delineated the primary structures of different families of receptors and channels in collaboration with Drs Nakanishi, Mishina and other co-workers. These structures included a neurotransmitter-gated channel (nicotinic acetylcholine receptor), voltage-gated channels (sodium channel and calcium channel), an intracellular membrane channel (calcium-release channel), and a G-protein-coupled receptor (muscarinic acetylcholine receptor). Expression of the cloned cDNAs produced functional receptors and channels. The structural basis for the function of neurotransmitter receptors and ionic channels was investigated by expression of wild-type and mutated cDNAs followed by electrophysiological analysis of the resulting products. The main findings obtained by Numa and co-workers contributed much to our understanding of the molecular basis of the functional alteration of the nicotinic acetylcholine receptor during skeletal muscle development, the identification of a channel-forming region of the nicotinic acetylcholine receptor, the identification of a voltage sensor segment responsible for activation of the sodium channel, the functional role of the skeletal muscle slow calcium channel in excitation-contraction coupling
327
17
-
SEPTEMBER 1992
JOURNALCLUB One of the most fashionable drugs in recent membrane traffic research has been brefeldin A. This drug causes the 'disappearance' of the Golgi complex, and the return of Golgi markers to the endoplasmic reticulum (ER) (for review see Ref. 1). The loss of a well-defined, compact Golgi structure has been observed previously, for example during mitosis 2, but the mechanism for Golgi disassembly is still not at all clear. Three recent papers 3-5 may shed light on the mechanisms involved in Golgi stability and maintenance. In one 3, a process indistinguishable from that observed after brefeldin A treatment was described upon overexpression of a putative ER salvage receptor. In the other two papers 4'5, Golgi disappearance was observed in cells expressing temperature-sensitive secretion-defective mutations in the End4 complementation group.
Membrane traffic between the ER and Golgi The ER represents the entry site of the secretory pathway. Secretory and membrane proteins are transferred from the ER to the Golgi during forward/ anterograde membrane traffic, whereas resident ER proteins or transportincompetent proteins are retained 6. A recycling pathway from the Golgi to the ER to retrieve any proteins that have escaped ER retention appears to be exaggerated by the drug brefeldin A1. This type of transport is termed reverse or retrograde membrane traffic, since it operates against the bulk flow7 of membrane and secretory proteins out of the cell. In order to maintain the structure of organelles along the secretory pathway, tight controls of forward and reverse traffic must exist. Brefeldin A perturbs the control of membrane traffic in a most profound fashion, most • probably by increasing retrograde while blocking anterograde transport, thus causing the disappearance of the Golgi complexL
Golgi appearance and disappearance In the electron microscope the Golgi complex has been shown to consist of several distinct sets of stacked cisternae connected by tubules 8 while in im© 1992, Elsevier Science Publishers, (UK)
Now you see it, now you don't: the Golgi disappearing act munofluorescence microscopy it is observed as a compact juxtanuclear structure in the centrosome region 9. The 'disappearance' of the Golgi is defined here as the loss of a compact juxtanuclear structure when observed by indirect immunofluorescence microscopy. After Golgi disappearance, staining of Golgi markers may be punctate, diffuse or in a well-dispersed reticulum, which is indistinguishable from the ER. Previous studies of the disassembled Golgi complex during mitosis and during treatment with phosphatase inhibitors such as okadaic acid have led to the identification of Golgi remnants 2,~°that represent the end point of Golgi disassembly and are thought to be the template for Golgi reassembly. This disassembly leads to a block in transport to and through the Golgi complex. Golgi dispersal is also seen in the presence of microtubule-disrupting drugs 11. Here, intact Golgi stacks disperse from their normal position clustered around the centrosome. There is little evidence that such a dispersal impairs intracellular transport through the Golgi to any great extent, though there may be some effects on exit from the Golgi in these cells. The final type of Golgi disappearance is observed in cells treated with the fungal metabolite brefeldin A1. In this case, the Golgi complex appears to be disassembled by the formation of tubules, which emanate from Golgi cisternae and fuse with the ER, eventually leading to the complete redistribution of Golgi markers and enzymes to the ER, where they can act on resident ER proteins. This also leads to a block in the secretory pathway, probably at the level of export from the mixed ER-Golgi compartment.
Overexpression leads to Golgi disappearance Hsu e t al. 3 looked at the effects of overexpression of a human homologue of the ERD-2 ER salvage receptoP 2 and a closely related protein, which they named ELP-1. Using transient expression systems they looked for cells expressing varying levels of ELP-1 or ERD-2. In cells that expressed high levels of ELP-1 or ERD-2 an ER-type staining pattern was observed in immunofluorescence, whereas in the cells expressing lower levels of ELP-1 or ERD-2 a compact Golgi-like distribution was observed. Double-labelling experiments with a general Golgi marker, lentil lectin, showed that when ELP-I was found in an ER-like pattern, the lectin was found to stain in a similar fashion, rather than having its usual compact Golgi distribution. This redistribution was reminiscent of the changes induced by the drug brefeldin A. One of the first known effects of brefeldin A is the redistribution of [3-COP, a non-clathrin-coated vesicle coat protein ~3, to the cytosol. When the distribution of [~-COP was examined in cells overexpressing ELP-1 it was found to have become diffuse. Further effects of the overexpression of ELP-1 were a defect in secretion of cotransfected lysozyme and the induction of Golgitype processing of a cotransfected ER glycoprotein. All of these findings are similar to the known effects of brefeldin A. In order to explain why the overexpression of a putative salvage receptor induces a brefeldin A-like phenotype the authors propose a model in which overexpression of ELP-1 leads to the enhancement of retrograde traffic from the Golgi to the ER due to localized uncoating of Golgi membranes which contain ELP-1. If a high concentration of the salvage receptor leads to the release of Golgi coat proteins and the uncoated vesicles and tubules produced can participate in retrograde, but ~not anterograde transport between the ER and Golgi complex, this would lead to an imbalance in traffic between the ER and the Golgi and finally to a steadystate redistribution of Golgi compo, nents to the ER (Fig. 1).
325
TIBS 17 - SEPTEMBER 1992
(a)
¢._ ~ b q
-
~
(c)
(b) ~oGolgi
~O
.) O Golgi
C
~ o ~--
o Golgi ~-~ remnants?
ELP-I@?
I i•End4@?
Brefeldin A@ ..... End4EQ Brefeldin A@
Brefeldin A@
ER
)C
~
~
~
ER-GoIgi
~ Figure 1 Forward and reverse traffic between the ER and Golgi. (a) At steady state, in the absence of drug or mutant induction, forward and reverse traffic between the ER and Golgi are balanced, which maintains the well-defined structure of the Golgi complex. Separate ER and Golgi coated vesicles probably mediate forward traffic, while return traffic is thought to be via uncoated vesicles. Functional End4 protein may be involved in mediating transport from the ER 'to the Golgi. (b) A short time after the addition of brefeldin A or after mutant induction, retrograde transport via uncoated elements from the Golgi to the ER is enhanced while forward traffic is blocked, possibly due to inhibition of coat protein binding. Overexpression of ELP-1 might assist in the increase in retrograde traffic, while lack of functional End4 protein might block export from the ER. Brefeldin A induces the formation of retrograde tubules which are thought to mediate reverse traffic from the Golgi to the ER and at the same time seems to inhibit forward traffic. All of these effects might be mediated by an inhibition of coat protein binding to ER or Golgi membranes. (¢) After longer times in the presence of drug or after mutant induction a new steady state is reached. The Golgi has disappeared to form a miXed Golgi-ER organelle, possibly leaving Golgi remnants that are incapable of participating in membrane traffic. Lack of functional End4 protein or the presence of brefeldin A continues to block export from the ER. This condition can be reversed upon removal of brefeldin A or return to the permissive temperature.
Mutations leading to Golgi disappearance In the other two papers in which Golgi disappearance was observed the effects of temperature-sensitive secretion-defective mutations were examined. Zuber et al2 looked at a temperature-sensitive mutant cell line, DS28-6, using immunofluorescence and electron microscopy. They observed the redistribution of Golgi markers from a compact juxtanuclear cisternal structure to a more diffuse distribution. They also provided some evidence that at the non-permissive temperature some Golgi markers were found in the ER. Looking at another mutant cell line, V.24.1, in the same complementation group (End4), Kao and Draper 5 also found that typical Golgi morphology was lost at high temperatures. However, Golgi enzymes did not appear to become functional components of the ER and immunofluorescence did not reveal Golgi markers in an ER staining pattern. They also examined the site of the block in secretion which appeared to be at the exit site from the intermediate compartment 14,15 between the ER and Golgi. Golgi disassembly appeared to be assisted by an intact microtubute system (a phenomenon also observed during brefeldin A action) and by continuing forward traffic through the Golgi.
326
Viewing the structure of the Golgi and the ER as the products of a steadystate balance between forward and reverse traffic between the two compartments the findings in these two papers may not be incompatible. If the intermediate compartment is morphologically similar to the ER, but its contents are biochemically distinct from those of the ER, EM morphology could localize Golgi components to the ER while Golgi enzymes could n o t act on bona fide ER markers.
Models of Golgi stability The papers discussed above raise some important questions about'-the nature of controls governing membrane traffic between the ER and the Golgi complex. How is the structure of the Golgi complex maintained in most ceils in the absence of drug or mutant perturbations? How can overexpression of one protein and inactivation of another lead to very similar phenotypes? How similar are these two phenotypes? What might be the function and distribution of the proteins corresponding to the End4 complementation group, and does it interact with the salvage receptors or Golgi coat proteins? In the End4 mutants at the non-permissive temperature, what is the effect on the salvage receptors and on the coat proteins and
how rapid is the onset of any affects found? Figure 1 outlines some possible sites of action and control of the proteins involved in maintaining the balance between forward and reverse traffic between the ER and Golgi. Blocking exit from the ER or increasing return from the Golgi could both lead to the establishment of a combined ER-Golgi compartment. The model can be increased in complexity when we consider that there may need to be an intermediate compartment between the ER and Golgi. This would probably then be the site from which recycling would usually occur, and in the End4 mutant appears to be the site where secretory proteins are blocked. The level of interaction between the ER and intermediate compartment and between the Golgi and the intermediate compartment is currently under investigation in several laboratories, and their findings will probably refine our understanding of the End4 les'ion. Research into intracellular transport is very active at the moment and candidate proteins involved in membrane traffic are coming to light at an astonishing rate. Our picture of the processes involved in intracellular membrane traffic and in the maintenance of stable organelles within the context of
TIBS 17 - SEPTEMBER 1992 very extensive membrane flow through the pathway is becoming clearer. It will be important to continue studies using the combined approaches of molecular genetics, cell biology on intact cells and cell-free assays of intracellular transport to continue the rapid advances in our knowledge of these mechanisms. References 1 Klausner, R. D., Donaldson, J. G. and LippincottSchwartz, J. (1992) J. Cell Biol. 116, 1071-1080 2 Lucocq, J. M. and Warren, G. (1988) EMBO J.
6, 3239-3246 3 Hsu, V. W., Shah, N. and Klausner, R. D. (1992) Cell 69, 625-635 4 Zuber, C. et al. (1991) Proc. Natl Acad. Sci. USA 88, 9818-9822 5 Kao, C-Y. and Draper, R. K. (1992) J. Cell Biol. 117,701-715 6 Pfeffer, S. R. and Rothman, J. E. (1987) Annu. Rev. Biochem. 56, 829-852 7 HuRley, S. M. and Helenius, A. (1989) Annu. R~v. Cell Biol. 5, 277-307 8 Rambourg, A. and Clermont, Y. (1990) Eur. J. Cell Biol. 51, 189-200 9 Burke, B. et al. (1982) EMBO J. 1, 1621-1628 10 Lucocq, J., Warren, G. and Pryde, J. (1991) J. Cell Sci. 100, 753-759 11 Ho, W. C. et al. (1989) Eur. J. Cell Biol. 48,
250-263 12 Semenza, J. C., Hardwick, K. G., Dean, N. and Pelham, H. R. B. (1990) Cell 61, 1349-1357 13 HuRley, S. M. (1991) Trends Biochem. Sci. 16,
165-166 14 Schweizer, A. eta/. (1990) Eur. J. Cell Biol. 53,
185-196 15 Saraste, J. and Svensson, K. (1991) J. Cell Sci.
100, 415-430
STELLA M. HURTLEY Department of Biochemistry,Universityof Edinburgh Medical School, Hugh Robson Building, GeorgeSquare, Edinburgh,UK EH8 9XD.
'z
OBITUARY Shosaku Numa, Professor, Department of Medical Chemistry and Department of Molecular Genetics, Kyoto University Faculty of Medicine, Kyoto, Japan, passed away on February 15, 1992, about a month and a half before his formal retirement and after a nearly threeyear struggle with colon cancer and metastasis. Professor Numa was born on February 7, 1927, in Wakayama, Japan. In 1952, he received his MD from Kyoto University, and after having spent about 12 months in internship, he entered the First Department of Internal Medicine, Kyoto University Faculty of Medicine, for clinical training. However, he was not too happy about the quality of research in clinical medicine and in 1956 received a Fullbright Fellowship to work at the Department of Biological Chemistry, Harvard Medical School, Boston, where he carried out analytical work on serum lipoproteins under the guidance of Professor Oncley. In 1958, he moved to the Max-Planck-Instit0t fiir Zellchemie, Munich, as an Alexander yon Humboldt stipendiat and started to work on enzymology and metabolic regulation of lipids under the guidance of Professor F. Lynen. Three years later, he returned to Japan and was appointed an instructor in the Department of Medical Chemistry, Kyoto University Faculty of Medicine, and was soon promoted to associate professor. While continuing his own project on lipid metabolism, he also collaborated with me and made a large contribution towards the elucidation of the metabolic pathway of tryptophan in mam© 1992, Elsevier Science Publishers, (UK)
Shosaku Numa 1927-1992
mals and microorganisms. In 1963, he took a leave of absence to resume collaboration with Lynen, and in 1968, returned to Kyoto to become Professor in charge of the 2nd Department of Medical Chemistry. During the next ten years or so, he continued to work on lipid metabolism and, together with a large number of collaborators, published many important papers concerning the ,enzymes involved in fat metabolism, especially acetyl-CoA carboxylase, which included
their structures and regulatory mechanisms. In the early 1980s, his interest gradually shifted to the application of molecular biology techniques to the elucidation of the primary structure of neurotransmitter receptors and ionic channels. Professor Numa then cloned and sequenced the cDNA and delineated the primary structures of different families of receptors and channels in collaboration with Drs Nakanishi, Mishina and other co-workers. These structures included a neurotransmitter-gated channel (nicotinic acetylcholine receptor), voltage-gated channels (sodium channel and calcium channel), an intracellular membrane channel (calcium-release channel), and a G-protein-coupled receptor (muscarinic acetylcholine receptor). Expression of the cloned cDNAs produced functional receptors and channels. The structural basis for the function of neurotransmitter receptors and ionic channels was investigated by expression of wild-type and mutated cDNAs followed by electrophysiological analysis of the resulting products. The main findings obtained by Numa and co-workers contributed much to our understanding of the molecular basis of the functional alteration of the nicotinic acetylcholine receptor during skeletal muscle development, the identification of a channel-forming region of the nicotinic acetylcholine receptor, the identification of a voltage sensor segment responsible for activation of the sodium channel, the functional role of the skeletal muscle slow calcium channel in excitation-contraction coupling
327
