A Study of the Protein Secretory Pathway of

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Fungal Genetics and Biology 32, 55–65 (2001)doi:10.1006/fgbi.2000.1245, available online at http://www.idealibrary.com on

Aspergillus niger Using a Glucoamylase–GFPusion Protein

Vahid Khalaj, Jayne L. Brookman, and Geoffrey D. Robson1

School of Biological Sciences, University of Manchester, 1.800 Stopford Building,Manchester M13 9PT, United Kingdom

Accepted for publication December 12, 2000; published online February 9, 2001

Khalaj, V., Brookman, J. L., and Robson, G. D. 2001. the vacuoles, indicating retargeting of the fusion pro-

A study of the protein secretory pathway of Aspergil-lus niger using a glucoamylase–GFP fusion protein.Fungal Genetics and Biology 32, 55– 65. The effectof various treatments that block protein secretion wasvisualized in Aspergillus niger using a strain express-ing a glucoamylase–GFP fusion protein. Cold shockcaused the retention of the fusion protein in a reticu-late network (ER) with brighter nodes that may repre-sent Golgi bodies. Treatment of germlings with brefel-din A (BFA) also initially caused accumulation withinthe ER but prolonged exposure led to the formationand targeting of the fusion protein to vacuoles from theER. Disruption of actin with cytochalasin A initially ledto a faint diffuse accumulation and ultimately to theformation of aggregated bodies which were not vacu-oles, suggesting that the actin cytoskeleton is impor-tant in secretory vesicle transport. Disruption of mi-crotubules with nocodazole led to hyperbranching butdid not cause intracellular accumulation, suggestingthat microtubules play a role in directing vesicle trans-port rather than vesicle movement per se. Treatment ofregenerating protoplasts confirmed that BFA and cy-tochalasin but not nocodazole inhibited protein secre-tion. When germlings were subjected to carbon star-vation, vacuolation was rapidly initiated throughout thehyphae and GFP fluorescence was visible in some of

1 To whom correspondence should be addressed. Fax: 44 161 275656. E-mail: Geoff.Robson@man.ac.uk.

1087-1845/01 $35.00Copyright © 2001 by Academic PressAll rights of reproduction in any form reserved. 55

tein from the secretory pathway to the vacuoles.

Index Descriptors: Aspergillus niger; protein secre-tion; glucoamylase; GFP; brefeldin A; cytochalasin;nocodazole; actin; microtubule; golgi; ER.

The filamentous fungi have a long history of commercialexploitation for the production of extracellular proteins dueto their high secretory capacity (Lowe, 1992; Oxenboll, 1994)and are attractive hosts for the commercial production ofheterologous proteins (Archer, 2000). However, despite theirimportance, our knowledge of the molecular basis of thesecretion process in filamentous fungi is limited (Peberdy,1994; Gouka et al., 1997; Archer and Peberdy, 1997). Nu-merous ultrastructural studies (Grove and Bracker, 1979;Howard and Aist, 1979; Howard, 1981; Roberson and Fuller,1988) in different fungi have shown organized endomem-brane systems within the hyphae but the precise spatialorganization of these membranes appears to vary betweendifferent fungal genera. Rothman (1994) has proposed acommon molecular mechanism for vesicle traffickingthroughout eukaryotes, although it is not clear whether thesepathways are present in the filamentous fungi. For example,whereas there is no direct evidence of vesicle traffickingbetween the ER and the Golgi, Veldhuisen et al. (1997) havecloned a homologue of the yeast small GTPase, SAR1, inAspergillus niger, involved in vesicle budding and Whittakeret al. (1999) have cloned sod VIC, an a-COP-related gene inAspergillus nidulans, providing indirect evidence of thesepathways in filamentous fungi.

A number of novel trafficking pathways between differ-

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56 Khalaj, Brookman, and Robson

ent endomembrane systems have been described in highereukaryotes using transport inhibitors such as brefeldin A(BFA) and nordihydroguairetic acid (NDGA). For exam-ple, retrograde vesicle transport from the Golgi apparatusto the ER has been demonstrated by the use of BFA(Klausner, 1992; Dinter and Berger, 1998) and NDGA(Tagaya et al., 1996; Fujiwara et al., 1998; Drecktrah et al.,1998) in eukaryotic cells.

Recently, GFP–chimeras have been used in filamen-tous fungi to provide in vivo visualization of specific or-ganelles and processes including nuclear migration andprotein secretion (Sulmann et al., 1997; Fernandes-Aboluset al., 1998; Gordon et al., 2000a,b). We have used an A.niger strain expressing a GFP–glucoamylase fusion pro-tein to study the influence of a number of protein trans-port and cytoskeletal inhibitors on the protein secretorypathway. This study presents the first in vivo visualizationf the spatial and temporal organization of the fungalndomembrane system following chemical perturbation.

MATERIALS AND METHODS

Fungal Strain and Plasmids

Wild-type A. niger N402 (cspA1 derivative of ATCC9029; Bos et al., 1988) was used for cotransformation with

lasmids Pgla–GLA499::sGFP (Gordon et al., 2000a) andAN7.1 (Punt et al., 1992).

Growth and Culture Conditions

A. niger strains were grown and maintained on potatoextrose agar (Oxoid). For microscopy, spores of the trans-ormant were grown on coverslips at 30°C under a thiniquid layer of modified Vogel’s medium (Vogel, 1956;

odified by substitution of 10 g L21 maltodextrin and 0.5 gL21 glucose for sucrose). For starvation experiments,germlings grown on coverslips in modified Vogel’s me-dium were briefly washed in phosphate-buffered saline(PBS) and then transferred to the same medium lackingmaltodextrin and glucose.

Protoplasts were prepared by gentle agitation of myceliaat 30°C in a lytic solution containing 5% (w/v) Glucanex(Novo Nordisk Ferment Ltd., Switzerland) and 0.6 M KClfor 3 h. Protoplasts were separated by filtration throughfour layers of sterile lens tissue, washed twice with 0.6 MKCl, transferred to modified Vogel’s liquid medium con-

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incubated for 2 h prior to chemical treatment. BFA (Sigma)was prepared as a 10 mg ml21 solution in methanol, NDGA(Sigma) as a 0.5 M solution in methanol, cytochalasin A(Sigma) as a 2 mg ml21 solution in dimethyl sulfoxide(DMSO), and nocodazole (Sigma) as a 4 mg ml21 solution inDMSO. Chemicals were added directly to the mycelia at anappropriate dilution in modified Vogel’s medium or to pro-toplasts at an appropriate dilution in modified Vogel’s me-dium containing 0.6 M KCl and 10 mM Tris–HCl (pH 7.2).

Transformation Procedure

A. niger N402 cultures were grown for 16–18 h in maltextract broth (Oxoid) and protoplasts were prepared as de-scribed above. Cotransformation with GLA499::sGFP vectorand pAN7.1 was mediated by polyethylene glycol (Punt andVan den Hondel, 1992). Protoplasts were regenerated onmodified Vogel’s medium containing 1.2 M sorbitol, 1% (w/v)glucose, and 200 mg ml21 hygromycin. Transformants con-taining the GFP construct were screened by fluorescencemicroscopy. Integration and expression of the construct wasconfirmed by standard Southern analysis (Southern, 1975)and Western blotting (Sambrook et al., 1989) using anti-GFPantibody (Clontech).

Microscopy

Mycelia on coverslips were viewed directly by a LeicaDMRXA microscope using standard FITC filters for GFPand standard DAPI filters for both DAPI-stained nucleiand CMAC–Arg (Molecular Probes)-stained vacuoles,with FluoTAR 3100 and 360 lenses. Images were col-ected using the IPLAB spectrum program and trans-erred to Adobe Photoshop version 5.0. To observe theuclei, mycelia were stained with 1 mg ml21 DAPI (Sigma)

for 0.5–1 min at room temperature. For vacuole staining,mycelia were rinsed briefly in PBS, covered with a 1:100dilution of a 5 mg ml21 CMAC–Arg solution in DMSO for15 min at room temperature, and washed briefly in PBSbefore being observed under the microscope.

RESULTS

Expression of GLA::GFP in A. niger

GLA::GFP expression was observed after 16 h growth inliquid medium and fluorescence was observed localized in

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57Protein Secretory Pathway in Aspergillus

the cell walls and septa with no intracellular accumulation(Fig. 1a, bottom) as previously demonstrated by Gordon etal., (2000a,b). Measurements of the specific growth rate inliquid culture and of the colony radial growth rate on solidmedia revealed no significant difference between the pa-rental strain (N402) and the cotransformant.

Accumulation of GFP Fluorescencenduced by Cold Shock

Figure 1b (lower) shows the appearance of GFP fluo-rescence in a reticular network with brighter nodes withinthe hyphae following 2–4 h of cold shock (4°C). Incuba-tions for more than 12 h under cold-shock conditionsresulted in a diminished level of fluorescence in thesestructures. Recovery at room temperature was associatedwith a loss of reticular fluorescence after 4–6 h and apattern of distribution identical to that observed prior tocold treatment (results not shown).

Effect of BFA and NDGA Treatment on theattern of GFP Fluorescence

The fate of the GLA::GFP fusion protein followingtreatment with 10–100 mg ml21 BFA was monitored over

FIG. 1. Pattern of GLA::GFP fluorescence in germlings of A. niger gb). Upper panel represents phase contrast images of the lower panel flu

mm; bar in (b), 25 mm.

a 16-h period. Careful examination of different popula-tions of hyphae revealed that only a proportion (ca. 30%)were affected at concentrations less than 50 mg ml21,

hereas 100 mg ml21 BFA was sufficient to induce aneffect in the majority (.90%) of hyphae. This higherconcentration was therefore used for subsequent treat-ments.

During the first 1 h following BFA treatment, a branch-ing reticulate fluorescent network which increased in in-tensity with time was observed throughout the hyphae(Fig. 2a) and it surrounded DAPI-stained nuclei (Fig. 2d).A similar pattern of fluorescence was also observed inBFA-treated protoplasts (results not shown). Subse-quently, after 3–6 h BFA treatment, dense vesicle-likebodies that varied in size (0.3–1 mm diameter; Fig. 3,right), gradually replaced the reticulate GLA::GFP fluo-rescent network. CMAC–Arg staining showed that themajority of these structures were vacuoles, although theywere not clearly visible under phase microscopy at thisstage (Fig. 4). After 6–8 h of exposure to BFA, largervesicles containing GFP (2–3 mm diameter) appearedwithin the hyphae and after 8 h were the predominantstructures present, many of which were visible underphase microscopy (Fig. 5). These large spherical vacuoleswere rarely present in untreated hyphae grown under

Vogel’s medium for 16 h at 30°C (a) and after transfer to 4°C for 4 ht images. Arrows in (b) indicate brighter punctate nodes. Bar in (a), 15

rown inorescen

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58 Khalaj, Brookman, and Robson

identical conditions. When treated hyphae were removedfrom the drug, growth recovered and was associated witha loss of intracellular fusion protein and the reestablish-ment of a phenotype identical to that observed prior totreatment (results not shown).

The presence of GFP fluorescence in the septa was alsoaltered by BFA treatment. In control samples and for 2 hafter addition of BFA, the majority of hyphae showed clearseptum fluorescence; however, loss of septal fluorescencewas evident in some hyphae after 2–4 h exposure to BFA(Fig. 5).

Cytochalasin A Treatment Inducesesicular Accumulation of GFPluorescence

To examine the role of actin in the trafficking of the GFPfusion protein, mycelia and protoplasts were treated with the

FIG. 2. GLA::GFP accumulation in germlings of A. niger treated withontrast image. The reticulate network (c) was seen to surround nuclei i

(c) and (d) show the positions of the nuclei.

FIG. 3. GLA::GFP accumulation in germlings of A. niger tre

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actin-depolymerizing agent cytochalasin A (CA). Examina-tion of cells after 1 h treatment with 80 mg ml21 CA showeda diffuse pattern of GFP fluorescence which became increas-ingly punctate with time. After 2–3 h exposure, GFP fluo-rescence was observed in discrete vesicular structures (30–50nm; Fig. 6a, right) which did not stain with CMAC–Arg (datanot shown). Treated protoplasts exhibited a similar pattern ofaccumulation (Fig. 6b, right). Treatment with CA, unlikeBFA, was not associated with a loss of septal fluorescence(Fig. 6a). Removal of the drug restored the phenotype to thatobserved prior to treatment and was associated with a loss ofintracellular fusion protein (results not shown).

Nocodazole Does Not Induce GFPccumulation

The microtubule depolymerizing agent nocodazolewas used to treat hyphae and protoplasts at a concen-

g ml21 BFA for 2 h in a reticulate network; (a) fluorescence; (b) phaseI-stained germlings (d). Bar in (a), 10 mm; bar in (c), 20 mm. Arrows in

ith 100 mg ml21 BFA for 6 h in vesicular bodies. Bar, 20 mm.

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59Protein Secretory Pathway in Aspergillus

tration of 25–100 mg ml21. Treatment of mycelia causedhyperbranching of the growing hyphae after 1–2 h (Fig.7). To confirm that nocodazole had induced microtu-

FIG. 4. GLA::GFP accumulation and CMAC–Arg staining in germlin(b) GLA::GFP; (c) CMAC–Arg staining. Bar, 10 mm.

FIG. 5. GLA::GFP accumulation in germlings of A. niger treated withArrows indicate the location of septa. Bar, 10 mm.

bule depolymerization, treated hyphae were stainedwith DAPI. Nuclei were visible in the older preexistinghyphae, but were absent from regions of the hyphae

niger treated with 100 mg ml21 BFA for 4 h. (a) Phase contrast image;

ml21 BFA for 8 h in large vacuoles. Some reticular staining is still visible.

gs of A.

100 mg

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Effect of BFA, Cytochalasin, and

60 Khalaj, Brookman, and Robson

which had grown after nocodazole treatment, confirm-ing that nocodazole had blocked nuclear division andmigration (data not shown). GFP fluorescence was vis-ible in the cell walls of newly formed hyphae followingnocodazole treatment and septal fluorescence was un-affected (Fig. 7).

FIG. 6. GLA::GFP accumulation in germlings (a) and protoplasts (b)of A. niger treated with 80 mg ml21 cytochalasin A for 3 h. Bar in (a), 20mm; bar in (b), 10 mm.

FIG. 7. Effect of treating germlings of A. niger with 100 mg ml21

accumulation. Bar, 30 mm.

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Nocodazole Treatment on ProtoplastRegeneration

When untreated protoplasts were regenerated in liquidmedium, GFP fluorescence began to appear around theprotoplasts after ca. 5–6 h, indicating secretion of theGLA::GFP fusion protein and regeneration of the cell wall(Fig. 8a). A similar pattern of fluorescence was also ob-served in the presence of nocodazole (Fig. 8b). However,for protoplasts treated with either BFA or CA, GFP flu-orescence accumulated within the protoplasts and no flu-orescence was observed around the outside, indicating nosecretion and no wall regeneration (Figs. 8c and 8d, re-spectively).

Glucose Starvation Leads toAccumulation of GFP Fluorescence inVacuoles

Fungal hyphae grown to mid-exponential phase in liq-uid culture and transferred to a carbon source-free me-dium were observed to undergo pronounced vacuolation,a response commonly observed when nutrients are ex-hausted (Klionsky et al., 1990; Paul et al., 1994; Papagianniet al., 1999). Subsequently, after ca. 2–3 h, GFP fluores-cence was observed to accumulate within some but not allof these structures (Fig. 9). When GFP fluorescence wasdetected, it was often restricted to the vacuoles nearest thetip, with a clear gradient of fluorescence intensity brightestat the hyphal apex (Fig. 9).

zole for 2 h. Note the hyperbranched phenotype but no intracellular

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61Protein Secretory Pathway in Aspergillus

DISCUSSION

In this study we attempted to block protein secretion inA. niger using different known chemical transport inhibi-tors and cold shock. To visualize events during secretionblocking we used a GLA::GFP fusion construct which wehave previously used to visualize protein secretion in A.

iger and to study intracellular accumulation in secretoryutants (Gordon et al., 2000a,b). In our previous study,

FIG. 8. Effect of drug treatments on protoplast regeneration of A.iger. Protoplasts were incubated for 3 h in osmotic medium at 30°C andhen treated for 2 h with either (a) no drug, (b) 100 mg ml21 nocodazole,c) 80 mg ml21 cytochalasin A, or (d) 100 mg ml21 BFA. Bar, 10 mm.

FIG. 9. Vacuolization and accumulation of GLA::GFP in res

N402) with plasmid Pgla–GLA499::sGFP containing thepyrG gene as a selectable marker. However, transformantswere consistently observed to germinate more slowly andto have a reduced growth rate compared to the parentalstrain N402, unless additionally supplemented with uri-dine (results not shown). As a consequence, for this study,we cotransformed the parental N402 strain withPgla–GLA499::sGFP and pAN7.1 (containing hygromycinas a selectable marker). The germination and growth rateof cotransformants were not significantly different fromthose of the parental N402 strain, and the levels of GFPfluorescence were greater than those of the pyrG trans-formants previously used (results not shown).

A short period of cold shock caused accumulation ofGFP in a reticular network with brighter punctate nodes.A similar network has been reported in A. nidulans and A.niger transformed with an ER-tagged GFP construct (Fer-nandez-Abalose et al., 1998; Gordon et al., 2000a) and inPisolithus tincorius following staining with the ER-Trackerdye (Cole et al., 2000). Thus, it appears that cold shockreduces post-ER secretion more quickly than it affectsprotein synthesis, leading to an accumulation of GFP inthe ER network, an observation also reported in bothmammalian (Presley et al., 1997; Roman and Garoff, 1985)and plant (Boevink et al., 1999) cells. However, longerperiods of cold shock led to disappearance of GFP fromthe ER, probably as a result of adaptation by the fungus tolow temperature. The brighter punctate nodes seen fol-lowing cold shock are similar to those identified as Golgibodies in P. tinctorius labeled with BODIPY–BFA andmay represent accumulation of GFP in the Golgi bodies inA. niger (Cole et al., 2000).

o 2 h glucose starvation in germlings of A. niger. Bar, 20 mm.

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The fungal metabolite BFA disrupts protein trafficking

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62 Khalaj, Brookman, and Robson

in eukaryotic cells and has been widely used to study thesecretory pathway in a wide range of organisms (Klausner,1992; Graham et al., 1993). Treatment of hyphae with

FA initially resulted in an accumulation of GFP in theame reticular network as that seen following cold shock.owever, in contrast, the brighter nodes observed follow-

ng cold shock treatment were not visible following BFAreatment. In mammalian cells, BFA treatment results inhe rapid arrest of secretion, accumulation of secretoryroteins in the ER, disassembly of Golgi apparatus, andhe mixing of the Golgi and ER membranes (Klausner et

al., 1992). However, in contrast, BFA treatment does notgenerally appear to promote Golgi disassembly in thefungi but does lead to ER proliferation, suggesting thatBFA inhibits ER to Golgi transport (Hayashi et al., 1982;

orre, 1990; Rupes et al., 1995; Bourett and Howard,996; Akashi et al., 1997; Cole et al., 2000). This wouldccount in this study for the lack of punctate fluorescencen BFA-treated cells, and the fluorescence staining of theR seen in this study as transport of the fusion protein

rom the ER to the Golgi would be blocked.In A. niger, after the initial accumulation of the GFP

usion protein within the ER, dense vesicle bodies ofarying sizes which increased in size with time and weressociated with a gradual decrease in the intensity of GFPuorescence within the ER were observed. Staining ofyphae with the vacuole-specific dye CMAC–Arg showedhat the majority of these bodies are vacuoles. This obser-ation strongly suggests that abnormal retention of pro-eins within the ER caused by BFA leads ultimately to thenitiation of vacuole formation and targeting of the ER-ccumulated proteins to the vacuoles. Cole et al. (2000)lso noted that BFA appeared to lead to the conversion ofhe tubular vacuolar network to aggregates of free vacuolesn P. tinctorius and Rupes et al. (1995) reported an in-rease in vacuolation in response to BFA in Schizophyllumommune, although they suggested that this might haveeen due to the onset of autolysis. In filamentous fungi,reatment with BFA and other agents that disrupt theecretory pathway leads to protein accumulation withinhe ER and initiation of an unfolded protein responseUPR) which includes up-regulation of a number of ER-ssociated chaperones and foldases (Jeenes et al., 1997;an Gemeren, 1997; Goller et al., 1998; Saloheimo et al.,999; Ngiam et al., 2000). The observations in this studyuggest that vacuolation and targeting of accumulated pro-eins within the ER is an important late-stage response ofhe UPR that allows protein degradation and recycling. Aecond inhibitor of vesicle transport from the ER to Golgi

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ver, NDGA was found to have little effect on A. niger,ith only very faint fluorescence observed after prolonged

8 h) exposure, suggesting that this agent is not effectiven fungi (results not shown).Polarized hyphal growth and protein secretion are de-

endent on the transport of secretory vesicles from theR/Golgi system toward the tip, and cytoskeletal compo-ents, including actin and myosin, are thought to have

mportant roles in this phenomena (Heath, 1995; McGold-ick et al., 1995; Steinberg, 1998; Geitmann and Emons,000). In Saccharomyces cerevisiae, secretory vesicles ac-umulated in actin-deficient mutants (Novick and Bot-tein, 1985), whereas in A. nidulans, incubation of myceliaith CA, an actin-depolymerizing agent, has been re-orted to lead to a reduction in protein secretion (Torralbat al., 1998). In the present study, the initial diffuse accu-ulation of GFP inside the hyphae after treatment withA suggests an initial accumulation of vesicles within the

ytoplasm and is consistent with the hypothesis that actinas a primary role in the movement of secretory vesiclesoward the apex. The vesicular structures that appear after–3 h of exposure were negative for CMAC–Arg stainingnd are not therefore vacuoles. These vesicular-like struc-ures may instead represent aggregations of secretory ves-cles.

Previous studies investigating the role of microtubulesn protein secretion and vesicle transport in fungi haveeen contradictory. Whereas some authors report thatgents causing microtubule depolymerization are associ-ted with a reduction in protein secretion (Jochova et al.,993; Delucas et al., 1993; Torralba et al., 1996), otherseport little affect or even an increase in secretion (Huf-aker et al., 1988; Pedregos et al., 1995). In this study, theajor effect of nocodazole as an antimicrotubule com-

ound was hyphal hyperbranching, a phenotype consistentith previous studies using microtubule-depolymerizinggents (Jochova et al., 1993; Caesar-Ton That et al., 1988).

In this study, nocodazole treatment did not result in anyintracellular accumulation of GFP inside the hyphae.Moreover, the formation of septa and cell wall fluores-cence in new branches, which arose after nocodazoletreatment, suggests that microtubules are not directly in-volved in vesicle trafficking per se, but rather are involvedin regulating the direction of vesicle flow. This observationsupports the findings of Reynaga-Pena et al. (1998), whereinhibitors of microtubules (but not actin) led to a loss ofgrowth directionality, suggesting that microtubules areimportant in controlling Spitzenkorper trajectory.

When filamentous fungi enter the stationary phase, a

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63Protein Secretory Pathway in Aspergillus

pronounced increase in vacuolation throughout hyphae isobserved as a result of nutrient deprivation (Paul et al.,1994; Ashford, 1998). Vacuoles in fungi have a number ofimportant roles, including macromolecular degradation,metabolite storage, and cytosolic ion and pH homeostasis(reviewed by Klionsky et al., 1990), and have been sug-ested to be equivalent to the endosomal–lysosomal sys-em in mammalian cells (Ashford, 1998). Entry into thetationary phase is associated with the onset of autolysisuring which cellular components, including the cell walls

n the latter stages, are hydrolyzed (McNeal et al., 1998;cIntyre et al., 2000). In the present study, vacuolationas induced within 2 h of glucose starvation and GFPuorescence was observed in a proportion of these. Thisonfirms that starvation stress induces a diversion of pro-eins within the secretory pathway, which were destinedor secretion to the vacuoles, although whether GLA::GFPs targeted to the vacuoles directly from the ER or fromhe Golgi is unknown. The observed gradient in GFPuorescence, with intensity decreasing in older vacuolesurthest from the tip, may reflect a gradient in GFPydrolysis, GFP in the youngest vacuoles having under-one less hydrolysis than GFP in the oldest. Alternatively,uch a gradient may reflect a higher concentration ofroteins in the ER nearest the tip than in the ER in olderegions.

ACKNOWLEDGMENT

The authors thank the Pasteur Institute of Iran for scholarship supportto V.K.

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H

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