Nonclassical protein sorting

(1993) Mol. Reprod. Dev. 36, 212-219 FASSLER, R., GEORCES-IABOUESE, E. and HIRSCH, E. (1996) Cm. Opin. Cell ho/. 8, 641-646 29 BERDITCHEVSKI, F., BAZZONI, G. and HEMLER, M. E. (1995) /. Biol. Gem. 270,17784-l 7790 30 WEBER, C., ALON, R., MOSER, B. and SPRINGER, T. A. (1996) I. Cell Biol. 134, 1063-l 073 31 CUO, M. W., WATANABE, T., MORI, E. and MORI, T. (1995) Zygote 3,65-73

BATES, P. (1996) Cell 86, l-3 DUNON, D., PIALI, L. and IMHOF, 8. A. (1996) Curr. Opin. Cell Biol. 8, 714-723 38 GILBERT, J. M., HERNANDEZ, L. D., BALLIET, J. W., BATES, P. and WHITE, J. M. (1995) /. Viral. 69, 7410-7415 39 ALLEN, C. A. and GREEN, D. P. L. (1995)). CellSci. 108, 767-777 40 PARRINCTON, I., SWANN, K., SHEVCHENKO, V. I., SESAY, A. K. and LAI, F. A. (1996) Noture 379, 364-368 41 CLARK, E. A. and BRUGCE, J. 5. (1995) Science 268,233-239

32

42

28

33 34 35

ROCHWERCER, L., COHEN, D. I. and CUASNICU, P. 5. (1992) Dev. Biol. 153, 83-90 HUANC, S., KAMATA, T., TAKADA, Y., RUGCERI, Z. M. and NEMEROW, G. R. (1996) /. Viral. 70,45024508 KING, S. L., CUNNINGHAM, J. A., FINBERG, R. W. and BERCELSON, 1. M. (1995) /. Viral. 69, 3237-3239 EVANDER, M. et 01. (1997) /. Viral. 71, 2449-2456

Eukaryotic cells are defined by their capacity to regulate intracellular functions by compartmentalization. This ability depends on numerous protein-sorting and targeting events. Three major paradigms of ‘classical’ protein sorting have been described to define the primary protein-targeting systems required for maintenance of basic organellar functions: direct membrane translocation, transport pores and vesicular protein trafficking. However, it is becoming increasingly apparent that many crucial protein-sorting events are mediated by the less-well-characterized ‘nonclassical’ targeting mechanisms. Classical

43 44 45

One of the classical paradigms involves direct transport through specifictranslocation channels-as occurs in protein translocation acrossthe endoplasmic reticulum (ER), chloroplast and mitochondrial membranes. There are many fine distinctions between these systems,but the basic mechanism common to this processis receptor-mediated recognition of a signalon the translocating polypeptide, followed by the passageof the polypeptide through an iontight proteinaceous channel, driven by a Pans-side Hsp70 homologue that binds to the incoming polypeptidel. As particular pathways such as mitochondrial targeting become increasingly well characterized, proteins that do not seemto follow the defined standard targeting pathways - asis the casefor cytochrome c - have been uncovered2. Furthermore, many additional mechanistic questions such ashow the channel is gated and how membrane proteins escapefrom the channel remain to be answered. A second paradigm involves transport of proteins through pores,suchasthoseinvolved in nuclear transport. Nuclear pore complexes areproteinaceousstructures that permit free diffusion of smallmoleculesand even small proteins. Transport of larger proteins and mRNAs between the nucleus and the cytosol is a selective processinvolving interaction between specific signalsand the transport machinery”. The third paradigm is vesicular transport. Proteins are transported from the ER lumen to destinations throughout the secretory system as well as to the (Vol.

7) June 1997

Copyright

Acknowledgements Work in the laboratory of J. M. W. was supported by a grant from the NIH. D. B. was supported in part by a grant from the Swiss National Science Foundation. M. C. and S. W. were supported by NIH training grants.

SCHWARTZ, M. A., SCHALLER, M. D. and GINSBERG, M. H. (1995) Annu. Rev. Cell Dev. Biol. 11,549-599 IWAO, Y. and FUJIMURA, T. (1996) Dev. i?iol. 177,558-567 MONCK, 1. R. and FERNANDEZ, J. M. (1992) /. Cell Biol. 119, 1395-l 404 LAWRENCE, Y., WHITAKER, M. and SWANN, K. (1997) Development 124,233-241

Nonclassical protein sorting Recent characterization

of the major protein-targeting

both yeast and mammalian

systems in

cells has provided detailed descriptions

of how cellular transport processes operate. Increasingly, however, novel protein-sorting

paradigms

trends in CELL BIOLOGY

36 37

mechanisms are being uncovered. These newly

discovered ‘alternative’

mechanisms ofprotein

sorting ensure

accurate delivery of numerous cellular constituents either to their resident compartment protein-degradation

or, in many cases, to the cellular machinery. Like the better characterized

%lassical’protein-sorting

systems, ‘nonclassical’

mechanisms involve both membrane translocation channels and vesicle-mediated current understanding

targeting through protein

transport. This review discusses our

of these nonclassical protein-sorting

pathways and their role in eukaryotic cells.

plasma membrane by vesicle-mediated reactionsG6. Vesicles containing lumenal cargo are formed at a donor membrane and then transported to an acceptor membrane. Subsequent fusion results in cargo delivery. Current research is directed at identifying the molecular components involved and elucidating their individual roles in vesicular protein sorting. These characterized protein-sorting pathways provide for the bulk of cellular organelle biogenesis. However, in many instances, cells require more specialized transport systemsand use‘nonclassical’pathways. For example, cellshave evolved various systems 0 1997

PII: SO962.6924(97)01050-7

Elsevier

Science

Ltd. All rights

reserved.

0962-8924/97/$17.00

The authors are in the Section of Microbiology, University of California, Davis, CA 95616, USA. E-mail: djklionsky @ucdavis.edu

225

protein-targeting machinery for this organelle (see Ref. 8 for a review). Although numerous proteinaceous components of the peroxisomal targeting machinery have been identified, at present, there is no consensus on the mechanism of peroxisomal import. Recent data demonstrating import of large substrates or folded oligomers suggest peroxisomal targeting is mediated either by a large pore as in nuclear transport or by a vesicular mechanism of protein localization9. Nonvesicular

pathways

One of the best-characterizedexamples of nonclassical transporters is the ATPbinding cassette(ABC) superfamily (see Ref. 10 for a review). Members of this family are present at many intracellular membranes and mediate the transport of numerous substrates,including drugs, FIGURE 1 ions, metabolites, peptides and proteins Nonclassical transporters. Nonclassical transporters are present at many subcellular membranes, (Fig. 1; Table 1). Important examplesof an including the endoplasmic reticulum (ER), peroxisomal, inner mitochondrial and plasma membranes. ABC transport systemarethe TAPl/TAl?? Except for MDRl (which is encoded as a single polypeptide), all of the transporters depicted here ABC family members, which transport function as either dimers or heterodimers, with each ‘half transporter’ consisting of six membranepeptides into the ERlumen for assembly spanning domains, and a conserved ATPase domain. Other nonclassical pathways include the targeting with moleculesof the classI major histoof KFERQ-containing proteins to lysosomes, the export of polypeptides destined for degradation from compatibility complex (MHC) and subthe ER via the Sec61 complex, and the secretion of galectin-1. sequent antigen presentation”. In the budding yeastSaccharomyces cerevisiae, the for disposing of misfolded proteins. Similarly, proteins nonclassicala-factor secretion systemrequires Ste6p, are selected for specific destruction when changing another ABC protein. So far, the only large proteins environmental conditions obviate the need for their known to be substratesfor this type of transporter are function. In addition, cellular events such as sporubacterial toxins such as haemolysin A, a 107-kDa lation require massive cellular remodelling, which is polypeptide. However, ABC proteins of unknown achieved in part by vacuolar uptake and degradation function exist in many intracellular membranes.Muof existing macromolecules’. Similar mechanisms tation of the gene encoding peroxisomal ABC proalso provide for the recycling of cellular components tein Pmp70 causesZellweger syndrome in humans. to promote survival during nutrient stress. This condition is characterized by the presence of Both vesicular and nonvesicular nonclassical proteinperoxisomal ghosts rather than fully formed organsorting pathways exist. For the most part, their mechaelles,consistent with a role for this protein in peroxinisms are not as well characterized as those that somal biogenesis.In S. cerevisiae,27 genesencoding mediate the classical sorting pathways. For this reaproteins with an ABC motif have been identified, inson, it is difficult to assess the relationship between dicating that this mode of transport is probably quite newly discovered protein-targeting mechanisms and widespread and may be responsiblefor the translothe classical sorting pathways. Some may be mechacation of many additional substrates. nistically similar to the classical pathways, whereas Mammalian cells secretevarious proteins into the others may define new protein-sorting paradigms. extracellular milieu by nonclassicalmechanisms.These One pathway for which the mechanism has yet to proteins, which include galectin-1, interleukin-la be elucidated is peroxisomal import. Substantial work and -j3, thioredoxin and basic fibroblast growth fachas gone into an examination of the signals used to tor, lack a signal sequenceand do not appearto enter direct proteins into the peroxisome as well as the the secretory pathway 12. Currently, it is unclear whether all the substratesfor nonclassical secretion usethe sameor different export pathways. Recently, TABLE 1 - SELECTED EURARYOTIC ABC TRANSPORTERS nonclassicalsecretion pathways were investigated in S. cerevisiaeby introducing the gene encoding mamName Cellular location Substrate malian nonclassicalsecretion substrategalectin-1. As in mammalian cells, galectin-1 was selectively seTAP1 /TAP2 ER membrane Antigenic peptides creted from yeast, even when transport through the MDRl Plasma membrane Amphiphilic cytotoxic drugs classical secretory pathway was inhibited13. In adSTE6 Yeast plasma membrane a mating factor dition, galectin-1 secretion did not require the Ste6p PMP70 Peroxisomal membrane Unknown ABC transporter, indicating that a novel nonclassical ATM 1 Mitochondrial inner membrane Unknown transport mechanism must be involved in the export YCFl Yeast vacuolar membrane Clutathione-S-conjugates of this polypeptidel”. Characterization of nonclassical 226

trends

in CELL

BIOLOGY

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1997

export mutants (rice) that are blocked in galectin-1 secretion will contribute to the identification of the molecular components involved in this process. Another nonclassical secretion pathway is export from the ER lumen (for a review, see Ref. 14). Recent studies indicate that ER degradation of mutant polypeptides is mediated by the proteasome. Genetic studies of a mutant form of carboxypeptidase Y that is degraded by the ER degradation pathway indicate that both ubiquitin-conjugating enzymes and proteasome subunits are involved in this process15. These results imply that the ER membrane possesses a transport system for exporting misfolded proteins to the cytosol. Additional insight into this pathway was provided by studies by Wiertz et al. on the mechanism of degradation of integral membrane MHC in cells infected with human cytomegalovirus (HCMV)16. They found that, when MHC degradation was stimulated either by HCMV infection or by addition of dithiothreitol in uninfected cells, the polypeptide was associated with Sec6lp before its ultimate degradation by the proteasome. Their results indicate that MHC first is transported into the ER through the Sec6lpcontaining translocon, then diffuses out of the translocon into the ER membrane, and finally, if folding and oligomerization requirements are not met, it reenters the Sec6lp complex and is ‘dislocated’ back to the cytosol for degradation. Sec6lp-mediated dislocation of soluble proteins destined for cytosolic degradation has not been demonstrated yet, but seems likely in light of these results. Additional components, such as molecular chaperones that might regulate the selection of polypeptides for degradation, have not been identified, but may include the ER-resident chaperone calnexin14. Finally, lysosomal membranes contain a specific transport system for the uptake of selected polypeptides for degradation in response to serum deprivation. Cytosolic proteins containing the KFERQ motif are transported selectively across the lysosomal membrane by a mechanism that appears to be similar to the membrane translocation systems of the ER and mitochondrial membranesl’. Transport of proteins by this pathway is driven by the hydrolysis of ATP and requires HSC73, a cytosolic protein of the Hsp70 family, as well as a Puns-side Hsp70’homologue16. Recently, the lysosomal membrane protein LGP96 has been identified as the receptor that binds the KFERQ motif, thereby selectively targeting proteins bearing this signal for lysosomal degradation19.

TABLE

2 - ALTERNATIVE

VACUOLAR-PROTEIN-DELIVERY

Pathway

Purpose

Signal

Macroautophagy

Degradative

Carbon

or nitrogen

Microautophagy

I

0

2 Autophagosome

API

FBPase

(d) Cytoplasm-to-vacuole targeting

L

2

0

API /;b)

Macroautophagy L9

FIGURE

2

Nonclassical and

mechanisms

one

biosynthetic

vacuole.

(a) In response

the vacuole

to glucose,

by 30-40-nm

degradation

(Vid)

or carbon,

cytosolic

nonselectively vacuole

of vacuolar nonclassical vesicles

pathwayzs.

in 400-900-nm

of peroxisomes is induced medium. uses many

Vesicular

selectively

the vacuolar

import

certain

vesicles

yeast

and

to the

(c) Sequestration membrane

glucose-containing

to the vacuole targeting

molecular

components

to and

for nitrogen

delivered

into

constitutively

to the

are sequestered

at the vacuole

are shifted

cytoplasm-to-vacuole of the same

proteins

cells are starved

organelles

by macroautophagy30.

(d) API is targeted

vesicle-mediated

is targeted

(P) by microautophagy when

degradative

FBPase through

and

Three target

(b) When

proteins

for degradation

delivery. pathways

by a

pathway

that

as macroautophagy.

pathways

Nonclassical vesicular transport mechanisms in mammalian cells are poorly characterized. The useof genetic approachesin yeast has increasedour understanding of these processes.Recent studies have focusedon alternative pathways used for vacuolar delivery. All yeast vacuolar localization pathways appear to be vesicle mediated; there is no evidence for protein translocation through a proteinaceous channel. Three processeshave been identified that share certain features: microautophagy, macroautophagy and cytoplasm-to-vacuole transport (Fig. 2). Microautophagy is usedfor the degradation of cytoplasmicproteins and organelles.This processinvolves uptake of cytoplasmic constituents directly at the vacuolar surface.Engulfment may occur by membrane invagination or through the formation of vacuolar protrusions. The best evidence for a distinction

PATHWAYS Specificity

Mutants

Maximal

rate

Refs

Bulk

apg, out, cvt

4% hr-’

29,32,34,36

Pdd gsa

?

37

?

21,27

vid apg, out, cvt

t,,, 30 min

25,26

t,!, 30 min

31,33,34,36

starvation Macroautophagy

Degradative

Glucose

addition

Specific

Microautophagy

Degradative

Glucose

addition

Bulk/specific

FBPase

Degradative

Glucose

addition

Specifc

Biosynthetic

Constitutive

degradation

Cytoplasm-to-vacuole

Specific

targeting

trends

in CELL

BIOLOGY

(Vol.

7) June

1997

227

between the micro- and macroautophagic pathways (see below) is found in Pichia pastotiszO. Under conditions where peroxisomes are no longer required, degradation of peroxisomes occurs through autophagy, but the specific mechanism differs depending upon nutrient conditions; glucose induces microautophagic uptake, whereas ethanol results in macroautophagy. The identification of mutants specifically defective in the glucose pathway, glucose-induced selective autophagy (gsa), supports the distinction between these pathways. The recent cloning of GSA1 revealed that this gene encodes the regulatory subunit of phosphofructokinase (PFKI; W. Dunn, pers commun.). However, the autophagy defect of a PFKl knockout can be rescued by both the wild-type PFKl gene and a mutated PFKl lacking the active site, suggesting that PFKI may modulate autophagy by a mechanism independent of its enzymatic activity (W. Dunn, pers. commun.). Fructose-1,6-bisphosphatase (FBPase) is a wellstudied gluconeogenic enzyme that is degraded upon the readdition of glucose to starved cells. There is considerable controversy regarding the mechanism of degradation -which may be vacuolar or proteasomedependent21-23. Indirect immunofluorescence of vacuolar import- and degradation-deficient (via) mutants suggests that FBPase is first sequestered within cytosolic vesiclesz4. Additional support for this finding comes from the recent purification of FBPase-containing cytosolic vesicles from wild-type cellszs. FBPase can also be detected at sites of invagination of the vacuolar membranez6, suggesting that both the Vid and the microautophagic pathway are used for its degradation. Because it occurs at the vacuole surface, microautophagy may appear mechanistically simpler than macroautophagy. Nonetheless, many questions remain concerning this process: first, if microautophagy involves the formation of membrane protrusions that engulf cytoplasmic contentsz7, what drives the deformation of the membrane? Are cytoskeletal elements involved? Second, if it takes place primarily by invagination, the direction of vesicle formation is into the extracellular space (vacuole lumen); again, what elements drive the membrane movement? Finally, in either case, how are specific substrates recognized and how is their binding/engulfment detected? Macroautophagy is also a degradative process, but the sequestration event does not occur at the vacuole membrane. Instead, double-membrane structures (autophagosomes) form in the cytosol and engulf proteins or organelles. The autophagosomes fuse with the vacuole, releasing a unilamellar vesicle inside the lumen. This autophagic body is degraded subsequently in a protease-B-dependent manner. Macroautophagy has been documented best through microscopy28-30, but, as with microautophagy, many questions remain to be answered. First, what is the origin of the autophagosome membrane? Second, how is the binding of the substrate detected? Finally, how is the completed autophagosome targeted to the vacuole? Both the ER and the Golgi have been proposed as source membranes, but this is problematic as these membranes are not likely to contain specific components such as V-SNARES that could direct the autophagosome to the vacuole. 228

The cytoplasm-to-vacuole targeting pathway (Cvt) is a biosynthetic process that targets at least one resident hydrolase, aminopeptidase I (API), to the vacuole31. Unlike autophagic uptake, import of API occurs constitutively under nutrient-rich, as well as starvation, conditions32. In addition, import of API is rapid, occurring with a half-time of 30-40 min (Ref. 33); this is faster and more complete than can be accounted for by bulk autophagy. Phenotypic and genetic analyses indicate that the Cvt and autophagic pathways overlap. First, in most of the mutants that were isolated based on defects in autophagy, apg34and n&35, API accumulates as a cytosolic precursor32,36; second, most cvt mutants that were isolated based on accumulation of precursor API are sensitive to nitrogen starvation, similar to the autophagy mutants32; and third, complementation studies suggest that large numbers of cvt/upg/aut mutants are allelic32,36. Recent studies indicate that precursor API is oligomerized rapidly into a dodecameric complex in the cytosol. It is then maintained as an oligomer as it completes the targeting process37. The transport of such a large oligomeric protein complex across a membrane such as the vacuole would almost certainly require a vesicular uptake mechanism, consistent with the genetic data that indicate that API transport and macroautophagy utilize many of the same molecular components. The vesicle-mediated vacuolar delivery pathways described above show some similarities. Both the degradative (FBPase and peroxisomal) and biosynthetic (API) pathways have a high degree of specificity and allow the import of particular organelles or proteins. Both microautophagy and macroautophagy result in the formation of vesicles that are released within the vacuolar lumen prior to breakdown. These uptake processes do, however, have substantial differences (Table 2). Bulk autophagy is induced upon starvation, whereas specific micro- and macroautophagy occur upon the addition of glucose or ethanol. API uptake occurs independent of nutritional signals, although the capacity can be stimulated upon starvation. The nonspecific uptake process is slow, having a half-time of at least 12 hours, Specific uptake, both degradative and biosynthetic, is rapid with half-times of 30-60 min26,32. The isolation and characterization of yeast mutants (apg34, auP, cvt31,36, vid 25,pdd 38) defective in the sequestration, vesicle fusion and/or breakdown steps, will allow a detailed understanding of these pathways. Concluding

remarks

Classicalsecretion and targeting pathways are by definition the bestcharacterized. They are apparently used by the greatest numbers of passengerproteins and for that reasonwere detected more easily. In most cases,it is not clear why alternative protein-sorting pathways are utilized. Perhaps these pathways are travelled more heavily than we realize; we just have not uncovered the majority of their substrates, or have not carefully examined the growth phasewhere they becomemore predominant. Alternatively, these pathways could be remnants of more-ancient proteinsorting mechanisms that are now utilized only in a trends in CELL BIOLOGY

(Vol.

7) June 1997

few specific cases. The nonclassical pathways outlined here serve a variety of roles. Elucidating the details of these systems has started to provide information about the tremendous complexity and diversity inherent in cellular physiology. References 1 SCHATZ, C. and DOBBERSTEIN, B. (1996) Science 271, 1519-1526 2 LILL, R. and NEUPERT, W. (1996) Trends Cell Biol. 6, 56-61 3 CijRLICH, D. and MAlTAJ, I. W. (1996) Science 271, 1513-l 518 4 ROTHMAN, J. E. and WEIIAND, F. T. (1996) Science 272,227-234 5 BEDNAREK, 5. Y., ORCI, L. and SCHEKMAN, R. (1996) Trends Cell Biol. 6, 468473 6 ARIDOR, M. and BALCH, W. E. (1996) Trends Cell Biol. 6, 31 S-320 7 ECNER, R. et ol. (1993) /. Biol. Chem. 268, 27269-27276 8 McNEW, J. A. and GOODMAN, J. M. (1996) Trends 6iochem. Sci. 21, 54-58 9 McNEW, J. A. and GOODMAN, J. M. (1994)). CellBiol. 127, 1245-1257 10 EGNER, R. et al. (1995) Membrane Protein Jronsport 2, 57-96 11 WILLIAMS, D., VASSILAKOS, A. and SUH, W-K. (1996) Trends Cell Biol. 6, 267-273 12 MUESCH, A. et ol. (1990) Trends Biochem. Sci. 15, 86-88 13 CLEVES, A. E., COOPER, D. N., BARONDES, 5. H. and KELLY, R. B. (1996) 1. Cell Viol. 133, 1017-l 026 14 BRODSKY, J. L. and MCCRACKEN, A. A. (1997) Trends Cell Biol. 7,151-156 15 HILLER, M. M., FINGER, A., SCHWEIGER, M. and WOLF, D. H. (1996) Science 273,1725-l 728 16 WIERTZ, E. J. H. J. et a/. (1996) Nature 384, 432-438 17 DICE, J. F. (1990) Trends Biochem. SC;. 15, 305-309 18 HAYES, 5. A. and DICE, J. F. (1996) /. Cell Biol. 132, 255-258

Alternative

trends in CELL BIOLOGY

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7) June 1997

pathways

19 CUERVO, A. M. and DICE, J. F. (1996) Science 273, Sol-503 20 TUlTLE, D. L. and DUNN, W. A., Jr (1995)/. Cell Sci. 108, 25-35 21 CHIANG, H-L. and SCHEKMAN, R. (1994) Nature 369,284 22 SCHORK, 5. M., BEE, G., THUMM, M. and WOLF, D. H. (1994) Nature 369,283-284 23 SCHORK, S. M., THUMM, M. and WOLF, D. H. (1995) /. Biol. Chem. 270,26446-26450 24 HOFFMAN, M. and CHIANG, H-L. (1996) Genetics 143, 1555-l 566 25 HUANG, P-H. and CHIANG, H-L. (1997) /. Cell Biol. 136,803-810 26 CHIANG, H-L., SCHEKMAN, R. and HAMAMOTO, S. (1996) /. Viol. Chem. 271,9934-9941 27 TUllLE, D. L., LEWIN, A. S. and DUNN, W. A., Jr (1993) Eur. 1. Cell Biol. 60, 283-290 28 BABA, M., OSUMI, M. and OHSUMI, Y. (1995) Cell Struct. funct. 20,465471 29 BABA, M., TAKESHIGE, K., BABA, N. and OHSUMI, Y. (1994) 1. Cell Biol. 124, 903-913 30 TAKESHIGE, K. et a/. (1992) 1. Cell Biol. 119, 301-311 31 HARDING, T. M., MORANO, K. A., SCOTT, 5. V. and KLIONSKY, D. 1. (1995) /. Cell Viol. 131, 591602 32 SCOTT, 5. V. et a/. (1996) Proc. Not/. Acad. Sci. U. 5. A. 93, 12304-l 2308 33 KLIONSKY, D. J., CUEVA, R. and YAVER, D. 5. (1992) I. Cell Viol. 119,287-299 34 TSUKADA, M. and OHSUMI, Y. I1 . 993) , FE81 lett. 333. 169-l 74 35 THUMM, ‘M. et a/. (1994) RBS Lett. 349, 275-280 36

HARDING, T. M., HEFNER-GRAVINK, A., THUMM, M. and KLIONSKY, D. J. (1996) j. Biol. Chem. 271, 17621-l 7624 37 KIM, J., SCOll, S. V., ODA, M. N. and KLIONSKY, D. J. 1. Cell Biol. (in press) 38 TITORENKO, V. I., KEIZER, I., HARDER, W. and VEENHUIS, M. (1995) I. Bacterial. 177, 357-363

of vacuolar

Acknowledgements We thank H-L. Chiana and W. Dunn fir communicating results before publication and Mark Havrilla for providing the cartoon reproduced below.

localization

229