TIBS 1 7 - J U N E 1 9 9 2
for constructive c o m m e n t s on t h e manuscript. Work in the Higgins laborat o r y is s u p p o r t e d by NSF grant DMB 9122048.
References 1 Drake, J. W. (1991) Annu. Rev. Genet. 25, 125-146 2 EIdredge, N. and Gould, S. J. (1972) in Models in Paleobiology(Schopf, T. J. M., ed.), pp. 88-115, Freeman, Cooper and Co. 3 Dorit, R. L., Schoenbach, L. and Gilbert, W. (1990) Science 250, 1377-1382 4 McClintock, B. (1984) Science 226, 792-801 5 Pato, M. L. (1989) in Mobile DNA (Berg, D. and Howe, M. M., eds), pp. 23-52, American Society for Microbiology
6 Shapiro, J. A. (1984) Mol. Gen. Genet. 194, 79-90 7 Shapiro, J. A. and Higgins, N. P. (1989) J. Bacteriol. 171, 5975-5986 8 Falconi, M. et al. (1991) New Biol. 3, 615-625 9 Hulton, C. S. J. et al. (1990) Cell 63, 631-642 10 May, G. et al. (1990) Mol. Gen. Genet. 224, 81-90 11 Sonti, R. V. and Roth, J. R. (1989) Genetics 123, 19-28 12 Reynolds, A. E., Mahadevan, S., LeGrice, S. F. and Wright, A. (1986) J. Mol. Biol. 191, 85-95 13 Hall, B. G. (1988) Genetics 120, 887-897 14 Kolter, R. (1992) Life and Death in Stationary Phase, Am. Soc. Microbiol. News 58, 75-79 15 Haselkorn, R. (1989) in Mobile DNA (Berg, D. and Howe, M. M., eds), pp. 735-742, American Society for Microbiology 16 Stragier, P., Kunkel, B., Kroos, L. and Losick, R.
(1989) Science 243, 507-512 17 Seifert, H. S. et al. (1988) Nature 336, 392-395 18 Scolnik, P. A. and Marrs, B. L. (1987) Annu. Rev. Microbiol. 41, 703-726 19 Heineman, J. A. and Sprague, G. F. (1989) Nature 340, 205-209 20 Cairns, J., Overbaugh, J. and Miller, S. (1988) Nature 335, 142-145 21 Cairns, J. and Foster, P. (1991) Genetics 128, 695-701 22 Hall, B. (1991) Proc. Natl Acad. Sci. USA 88,
5882-5886 23 Davis, B. D. (1989) Proc. Natl Acad. Sci. USA
86, 5005-5009 24 Stahl, F. W. (1988) Nature 335, 112-113 25 Shapiro, J. A. and Leach, D. (1990) Genetics
126, 293-299 26 Shakespeare, W. (1954) The Tragedy of Hamlet, Prince of Denmark I,v, p. 167, Folio Society
LETTERS Are molecular filters really necessary? The article by Hopkins 1 places great emphasis on the role of molecular filters in membrane protein trafficking. The molecular filters he refers to are supposed to be those originally proposed and documented by Brets'cher 2'3. However, the concept of the molecular filter as defined by Bretscher differs in at least one fundamental respect from that invoked by Hopkins. Bretscher proposed that the molecular filter was a device for preventing plasma membrane proteins from entering domains such as the coated pit~ so that these structures could preferentially internalize lipid for recycling and maintaining a directed lipid flow on the plasma membrane. This extreme concept was moderated to the idea that the coated pit operated to exclude most membrane proteins from the coated regions 3. Even in this attenuated form, however, the molecular filter was essentially a negative device for excluding membrane proteins from specific domains. In contrast, Hopkins employs the term almost entirely in the positive sense, that is, as a device for concentrating membrane proteins at specific sites, although this was not the sense in which it was 'originally proposed and documented by Bretscher'. Is our understanding of membrane protein sorting enhanced by the above concept, given the confusion surrounding the use of the term 'molecular filter'? In the positive filtering idea, it is unclear whether there is a special process involved or whether it is simply a different name for a well-known process,
in which case introducing a new term is confusing and unhelpful. As described by Hopkins, the coated-pit positive filter is thought to operate through a shell of adaptor complexes between the lipid bilayer of the membrane and the clathrin lattice, and that these complexes bind trafficking proteins. The underlying physicochemical principle leading to this chain of events is the well-known one of preferential affinity between macromolecules, which operates in a host of familiar processes such as the assembly of budding viruses, secretory granules and even organelles. It involves complex simultaneous interaction between arrays of macromolecules in a manner analogous to crystallization in solution. These latter processes exhibit the same selectivity in their assembly in that they focus specific molecular species (and exclude others) at a particular site, but it has never been necessary to refer to them as possessing a special filtering mechanism. Thus the use of the term filter in this context is unnecessary and confusing. This then leaves the question of the advantage of using the term in the context of the apparent exclusion of some components from the coated regions, i.e. the negative filter, as originally proposed. The evidence for the existence of selective exclusion is at best controversial 4 and could reasonably be treated with scepticism. However, even if this evidence is taken as accepted, is it necessary to invoke a special device to explain the observations? The central issue here is the state of the membrane in the coated regions. If the possibility that the membrane consists of closely packed proteins and iipids with some proteins
exhibiting a higher affinity for the membrane/adaptin/clathrin lattice is correct, it follows that others will be excluded for steric reasons, just as the majority of membrane proteins are excluded from the budding site of viruses. In such a system the exclusion observed does not involve any physicochemical principle other than the affinity of the membrane proteins for each other and/or the adaptin/clathrin lattice. In other words, there is no filter because the exclusion can be effected by the membrane molecules themselves. The consequence of these considerations is that the selectivity observed during protein trafficking along cellular pathways is the direct consequence of the selective affinity of proteins for each other at different points in the pathway. The exclusion of particular components is not the result of a filter, rather the simple consequence of the fact that there are spatial limitations at the assembly sites. The underlying principle is already well known in the secretory pathway, since secretory granules are believed to assemble in this way, i.e. condensation sorting 5. We have proposed that the selective assembly into supramolecular structures of varying stability also contributes to the sorting of resident luminal proteins such as reticuloplasmins in the ER6. Given these considerations, it seems to me that the term 'filtration' should be abandoned altogether in the context of selective membrane protein trafficking.
GORDON KOCH Laboratory of Molecular Biology, Hills Road, Cambridge, UK CB2 2QH.
211
for constructive c o m m e n t s on t h e manuscript. Work in the Higgins laborat o r y is s u p p o r t e d by NSF grant DMB 9122048.
References 1 Drake, J. W. (1991) Annu. Rev. Genet. 25, 125-146 2 EIdredge, N. and Gould, S. J. (1972) in Models in Paleobiology(Schopf, T. J. M., ed.), pp. 88-115, Freeman, Cooper and Co. 3 Dorit, R. L., Schoenbach, L. and Gilbert, W. (1990) Science 250, 1377-1382 4 McClintock, B. (1984) Science 226, 792-801 5 Pato, M. L. (1989) in Mobile DNA (Berg, D. and Howe, M. M., eds), pp. 23-52, American Society for Microbiology
6 Shapiro, J. A. (1984) Mol. Gen. Genet. 194, 79-90 7 Shapiro, J. A. and Higgins, N. P. (1989) J. Bacteriol. 171, 5975-5986 8 Falconi, M. et al. (1991) New Biol. 3, 615-625 9 Hulton, C. S. J. et al. (1990) Cell 63, 631-642 10 May, G. et al. (1990) Mol. Gen. Genet. 224, 81-90 11 Sonti, R. V. and Roth, J. R. (1989) Genetics 123, 19-28 12 Reynolds, A. E., Mahadevan, S., LeGrice, S. F. and Wright, A. (1986) J. Mol. Biol. 191, 85-95 13 Hall, B. G. (1988) Genetics 120, 887-897 14 Kolter, R. (1992) Life and Death in Stationary Phase, Am. Soc. Microbiol. News 58, 75-79 15 Haselkorn, R. (1989) in Mobile DNA (Berg, D. and Howe, M. M., eds), pp. 735-742, American Society for Microbiology 16 Stragier, P., Kunkel, B., Kroos, L. and Losick, R.
(1989) Science 243, 507-512 17 Seifert, H. S. et al. (1988) Nature 336, 392-395 18 Scolnik, P. A. and Marrs, B. L. (1987) Annu. Rev. Microbiol. 41, 703-726 19 Heineman, J. A. and Sprague, G. F. (1989) Nature 340, 205-209 20 Cairns, J., Overbaugh, J. and Miller, S. (1988) Nature 335, 142-145 21 Cairns, J. and Foster, P. (1991) Genetics 128, 695-701 22 Hall, B. (1991) Proc. Natl Acad. Sci. USA 88,
5882-5886 23 Davis, B. D. (1989) Proc. Natl Acad. Sci. USA
86, 5005-5009 24 Stahl, F. W. (1988) Nature 335, 112-113 25 Shapiro, J. A. and Leach, D. (1990) Genetics
126, 293-299 26 Shakespeare, W. (1954) The Tragedy of Hamlet, Prince of Denmark I,v, p. 167, Folio Society
LETTERS Are molecular filters really necessary? The article by Hopkins 1 places great emphasis on the role of molecular filters in membrane protein trafficking. The molecular filters he refers to are supposed to be those originally proposed and documented by Brets'cher 2'3. However, the concept of the molecular filter as defined by Bretscher differs in at least one fundamental respect from that invoked by Hopkins. Bretscher proposed that the molecular filter was a device for preventing plasma membrane proteins from entering domains such as the coated pit~ so that these structures could preferentially internalize lipid for recycling and maintaining a directed lipid flow on the plasma membrane. This extreme concept was moderated to the idea that the coated pit operated to exclude most membrane proteins from the coated regions 3. Even in this attenuated form, however, the molecular filter was essentially a negative device for excluding membrane proteins from specific domains. In contrast, Hopkins employs the term almost entirely in the positive sense, that is, as a device for concentrating membrane proteins at specific sites, although this was not the sense in which it was 'originally proposed and documented by Bretscher'. Is our understanding of membrane protein sorting enhanced by the above concept, given the confusion surrounding the use of the term 'molecular filter'? In the positive filtering idea, it is unclear whether there is a special process involved or whether it is simply a different name for a well-known process,
in which case introducing a new term is confusing and unhelpful. As described by Hopkins, the coated-pit positive filter is thought to operate through a shell of adaptor complexes between the lipid bilayer of the membrane and the clathrin lattice, and that these complexes bind trafficking proteins. The underlying physicochemical principle leading to this chain of events is the well-known one of preferential affinity between macromolecules, which operates in a host of familiar processes such as the assembly of budding viruses, secretory granules and even organelles. It involves complex simultaneous interaction between arrays of macromolecules in a manner analogous to crystallization in solution. These latter processes exhibit the same selectivity in their assembly in that they focus specific molecular species (and exclude others) at a particular site, but it has never been necessary to refer to them as possessing a special filtering mechanism. Thus the use of the term filter in this context is unnecessary and confusing. This then leaves the question of the advantage of using the term in the context of the apparent exclusion of some components from the coated regions, i.e. the negative filter, as originally proposed. The evidence for the existence of selective exclusion is at best controversial 4 and could reasonably be treated with scepticism. However, even if this evidence is taken as accepted, is it necessary to invoke a special device to explain the observations? The central issue here is the state of the membrane in the coated regions. If the possibility that the membrane consists of closely packed proteins and iipids with some proteins
exhibiting a higher affinity for the membrane/adaptin/clathrin lattice is correct, it follows that others will be excluded for steric reasons, just as the majority of membrane proteins are excluded from the budding site of viruses. In such a system the exclusion observed does not involve any physicochemical principle other than the affinity of the membrane proteins for each other and/or the adaptin/clathrin lattice. In other words, there is no filter because the exclusion can be effected by the membrane molecules themselves. The consequence of these considerations is that the selectivity observed during protein trafficking along cellular pathways is the direct consequence of the selective affinity of proteins for each other at different points in the pathway. The exclusion of particular components is not the result of a filter, rather the simple consequence of the fact that there are spatial limitations at the assembly sites. The underlying principle is already well known in the secretory pathway, since secretory granules are believed to assemble in this way, i.e. condensation sorting 5. We have proposed that the selective assembly into supramolecular structures of varying stability also contributes to the sorting of resident luminal proteins such as reticuloplasmins in the ER6. Given these considerations, it seems to me that the term 'filtration' should be abandoned altogether in the context of selective membrane protein trafficking.
GORDON KOCH Laboratory of Molecular Biology, Hills Road, Cambridge, UK CB2 2QH.
211
