Evaluation of the role of autophagy in fungal development and pathogenesis 3

The role of autophagy in carbohydrate catabolism and cell survival is discussed along with the specific functions of macroautophagy in fungal development and pathogenesis.1.. The majordi

136 First author publication as listed on page xi

Methods for Functional Analysis of Macroautophagy in Filamentous Fungi

Yi Zhen Deng,*Marilou Ramos-Pamplona,*and Naweed I Naqvi*

Contents

2 Methods for the Functional Analysis of Autophagy

mem-Methods in Enzymology, Volume 451 # 2008 Elsevier Inc ISSN 0076-6879, DOI: 10.1016/S0076-6879(08)03220-5 All rights reserved.

* Fungal Patho-Biology Group, Temasek Life Sciences Laboratory and Department of Biological Sciences, National University of Singapore, Singapore

295

and we summarize the methods that have been routinely used for monitoring macroautophagy in both yeast and filamentous fungi The role of autophagy in carbohydrate catabolism and cell survival is discussed along with the specific functions of macroautophagy in fungal development and pathogenesis.

1 Introduction

Autophagy is a highly conserved catabolic process in eukaryotes that isresponsible for organellar turnover, membrane recycling, and protein deg-radation in vacuoles/lysosomes Autophagy is induced in response to envi-ronmental stress or developmental signals during cellular differentiation(Besteiro et al., 2006; Noda and Ohsumi, 1998; Pinan-Lucarre et al.,

2003) Autophagy can act as a prosurvival signal or participate in grammed cell death, depending on the particular physiological conditions(Codogno and Meijer, 2005)

pro-There are three distinct classes of autophagy: macroautophagy, autophagy, and chaperone-mediated autophagy (CMA); the latter is selec-tively used to degrade cytosolic proteins containing a specific pentapeptideconsensus motif (Majeski and Dice, 2004; Salvador et al., 2000) Macro-autophagy and microautophagy (Reggiori and Klionsky, 2002) are consid-ered nonselective and thus have more degradative capacity The majordifference between macroautophagy and microautophagy is whether thedouble-membrane vesicles, autophasogosomes, sequester cytoplasmic pro-teins or organelles (macroautophagy) for delivery to the lysosome/vacuolefor degradation (Suzuki et al., 2001), or whether the cytoplasm is directlyengulfed into the vacuoles (microautophagy) (Mortimore et al., 1989).Besides general autophagy, some specific types of autophagy exist, such ascrinophagy (the activity of lysosomes related to the secretory pathway andendocrine functions;Glaumann, 1989), reticulophagy (degradation of ER;

micro-Bernales et al., 2007; Bolender and Weibel, 1973) and pexophagy dation of peroxisomes;Sakai and Subramani, 2000)

(degra-Thus far, 31 ATG genes (autophagy-related) have been characterized inSaccharomyces cerevisiae, which has led to a better understanding of the geneticand molecular regulation of autophagy (Kabeya et al., 2007; Klionsky et al.,

2003), particularly the formation of autophagy-associated vesicular ments, such as preautophagosomal structures (PAS), autophagosomes(cytosolic), and autophagic bodies (vacuolar) (Suzuki et al., 2001)

compart-1.1 Cellular functions of autophagy in filamentous fungi

Autophagy is reported to play a crucial role during differentiation of severalfilamentous fungi such as Podospora, Aspergillus, Colletotrichum, and Magna-porthe Autophagy-deficient mutants of Magnaporthe oryzae are nonpathogenicand show highly reduced asexual development (Liu et al., 2007; Veneault-Fourrey et al., 2006) Loss of autophagy-assisted programmed cell death in

atg8D appressorium is proposed to be responsible for the failure of penetratingthe host cuticle (Veneault-Fourrey et al., 2006) Furthermore, autophagy isinvolved in lipid body turnover and thus is essential for turgor generation andappressorium-mediated penetration (Liu et al., 2007) However, Colletotrichumgloeosporioides, with a related infection strategy as M oryzae, does not requireautophagic cell death for successful infection (Nesher et al., 2008) Surpris-ingly, infection structures/appressoria from a CLK1-deletion (an ortholog ofMgATG1) mutant in Colletotrichum lindemuthianum, are unable to penetratethe host cuticle (Dufresne et al., 1998), similar to the result from M oryzae In

S cerevisiae, loss of autophagy leads to failure of sporulation, sensitivity tonitrogen starvation, and increased pseudohyphal growth (Cutler et al., 2001;

Ma et al., 2007; Tsukada and Ohsumi, 1993) In A oryzae, autophagy isrequired for the differentiation of aerial hyphae and in conidial germination(Kikuma et al., 2006) In contrast to its function in fungi mentioned previ-ously, autophagy plays little or no role in the differentiation of the dimorphicyeast Candida albicans within the host tissue (Palmer et al., 2007) The atg9Dmutant in C albicans remains unaffected for yeast-hypha or chlamydosporedifferentiation, though it shows specific defects in autophagy and thecytoplasm-to-vacuole targeting (Cvt) pathway

In P anserina, autophagy is essential for sexual differentiation and celldeath by incompatibility It remains controversial whether autophagy exe-cutes a programmed cell death function or acts as a prosurvival response inPodospora (Dementhon et al., 2003, 2004; Pinan-Lucarre et al., 2003; Pinan-Lucarre et al., 2005) It was initially thought that autophagy acts as the cause

of cell death during incompatible interactions for it is induced when cells ofunlike genotypes fuse in P anserina (Dementhon et al., 2004; Pinan-Lucarre

et al., 2003) A recent study suggests that autophagy serves a prosurvival roleduring incompatibility, as loss of autophagy results in accelerated cell death(Pinan-Lucarre et al., 2005)

In this chapter, we present a technical review of the most frequently usedmethods to study autophagy in yeast and fungal species and focus on themethods that are useful to monitor the induction and rate of autophagy

in filamentous fungi The following protocols and methods have beenvalidated in the model fungus M oryzae and can be easily adapted andoptimized for use in other filamentous fungi of interest

2 Methods for the Functional Analysis of

Autophagy in Filamentous Fungi

2.1 Gene-deletion analyses to assess macroautophagy

in filamentous fungi

Generally, the one-step gene deletion strategy (schematized inFig 20.1) is usedfor disruption of requisite gene function in filamentous fungi For genedisruption of Magnaporthe ATG1 (MGG_06393.5), genomic DNA fragments

(about 1 kb each) representing the 50and 30flanks of the ATG1 open readingframe were amplified by PCR, and ligated sequentially so as to flank the ILV1cassette (confers resistance to sulfonyl urea) in pFGL385 to obtain plasmidvector pFGLatg1KO The pFGLatg1KO plasmid was transformed into wild-type M oryzae using Agrobacterium T-DNA-mediated transformation forhomology-dependent replacement of the ATG1 gene Gene-disruption con-structs can also be delivered into the fungal species of choice using electropo-ration of spheroplasts (Talbot et al., 1993; Vollmer and Yanofsky, 1986;Xoconostle-Cazares et al., 1996; Yelton et al., 1984) Transformants wereselected for resistance to chlorimuron ethyl (100 mg/mL; Cluzeau Labo,France) and correct gene-replacement confirmed by PCR analysis and South-ern blotting The primers used for amplifying the 1-Kb region at the 50- and

30-flank of the ATG1 gene were as follows: ATG1-5F (50-

GAGAGTGTTAAGCTTGGACGTACAGTAGG-TAATTGGT-30) The preceding protocol is validated for Magnaporthe butcan be easily optimized for other fungal species of choice, provided therequisite sequence information is available Other selectable marker cassettesfor transformation in fungi include hygromycin resistance (HPH1) orammonium-gluphosinate resistance (BAR) Gene targeting of the markercassette can also be achieved by providing homology within the codingsequence per se and need not be restricted to the flanking sequences asdescribed previously

2.2 Use of chemical inhibitors to investigate

autophagy in fungi

There are several chemical inhibitors of autophagy that are commerciallyavailable and routinely used in mammalian cells Although these inhibitorsblock autophagy, their effects are not entirely specific Wortmannin ( WM)

Figure 20.1 Schematic representation of a one-step gene replacement strategy for the ATG1 locus (MGG_06393.5) in Magnaporthe Solid bars and short open boxes represent coding regions and introns, respectively, whereas grey bars indicate the genomic flanks used as regions of homology for gene targeting Relevant restriction enzyme sites have been depicted and ILV1 refers to the sulfonyl urea-resistance cassette used to replace ATG1to create the atg1Dstrain.

is an inhibitor of PI3-kinase and blocks the induction of autophagy(Blommaart et al., 1997; Petiot et al., 2000) 3-methlyadenine (3-MA) isalso a classical inhibitor of the autophagic pathway (Seglen and Gordon,

1982) N-ethylmaleimide (NEM) inhibits several vesicular transport eventsand thus blocks the formation of autophagic vacuoles (Woodman, 1997).Such inhibitors can be potentially useful in studying autophagy in fungalsystems provided special caution is exercised in analyzing the results WM-treatment of vegetative mycelia (Fig 20.2) of Magnaporthe wild-type strainmimics the phenotype of an atg8D mutant, in which starvation fails toinduce autophagy (Deng and Naqvi, unpublished data)

Figure 20.2 Epifluorescence microscopy-based assessment of autophagosomes lia from the wild type or atg8D mutant were stained with MDC and imaged using epifluorescence microscopy MDC-stained wild-type mycelia pretreated with the autophagic-inhibitor Wortmannin (WM) prior to starvation, served as a negative control Scale bar denotes 5 mm Micrographs were pseudo colored using Photoshop Version 7.

1 Small amount of vegetative mycelia (approximately 50 mg of wetweight; scraped from a colony surface with inoculation loop) or conidia(ca 4  103) of wild-type and atg8D strains are cultured in 20 ml ofcomplete medium (CM; yeast extract 0.6%, casein hydrolysate 0.6%,sucrose 1%) for 48 h at 28C with gentle shaking (150 rpm) to obtainsufficient biomass

2 Wortmannin stock (1 mM, in DMSO) is added into the CM, to a finalconcentration of 200 nM The mycelia are treated with Wortmannin for

3 h at 28C with gentle shaking

3 Mycelia are harvested by filtration through sterile Miracloth chem, USA) and washed twice by filtration with sterile distilled water

(Calbio-4 A small amount of the freshly cultured mycelia is then inoculated into

20 ml of minimal medium lacking nitrogen [MM-N: 0.5 g/L KCl,0.5 g/L MgSO4, 1.5 g/L KH2PO4, 0.1% (v/v) trace elements, 10 g/Lglucose, pH6.5; (Talbot et al., 1993)] containing 2 mM PMSF, andgrown for 16 h with gentle shaking at 28C Please note that this step

is carried out to induce autophagy and there may not be a visible change

inter-et al., 1998), C albicans (Palmer et al., 2007), and P anserina (Dementhon

et al., 2004), starvation stress leads to enlarged vacuoles, which can beobserved by DIC optics Large punctuate perivacuolar structures or vesiclesinside the vacuolar lumen can also be visualized, indicating the formation ofautophagosomes or accumulation of autophagic bodies In the PodosporaDpspA mutant, which lacks the PSPA vacuolar protease, the accumulation

of autophagic bodies in the vacuolar lumen is even more striking and easilydetectable by simple microscopy observations (Dementhon et al., 2003).Fluorescence microscopy is another method to monitor the induction ofautophagy N-terminal tagging of Atg8 with a fluorescent protein such asGFP or RFP helps in epifluorescent detection of autophagosomes and hasbeen used in several filamentous fungi such as A oryzae (Kikuma et al.,

2006), P anserina (Pinan-Lucarre et al., 2005), and M oryzae (Deng andNaqvi, unpublished results) An advantage of utilizing GFP- or RFP-taggedAtg8 is that the extent of autophagosome formation correlates well with theamount of GFP/RFP-Atg8PE (Kabeya et al., 2000), so that the induction of

autophagy can be easily quantified by Western blotting using commerciallyavailable anti-GFP/RFP antibodies.

Transmission electron microscopy (TEM) is the gold standard for structural investigation of autophagy-associated membrane compartments

ultra-in filamentous fungi such as P anserultra-ina (Pinan-Lucarre et al., 2003),

A oryzae (Kikuma et al., 2006), and M oryzae (Liu et al., 2007; Fourrey et al., 2006) Fig 20.3 depicts the TEM analysis of the vacuolarlumen in the wild type and an autophagy-deficient mutant (atg8D) ofMagnaporthe

Veneault-Procedure

1 Fresh conidia (4  103) or small amounts of mycelia (scraped from acolony using inoculation loop; about 50 mg) from the wild-type oratg8D strain in Magnaporthe are grown in 20 ml of CM for 48 h at 28C

with gentle shaking (150 rpm)

2 Mycelia are harvested by filtration through Miracloth and washedthoroughly using sterile distilled water

3 Washed mycelia from individual strains are grown in liquid MM-Nmedium [0.5 g/L KCl, 0.5 g/L MgSO4, 1.5 g/L KH2PO4, 0.1% (v/v)trace elements, 10 g/L glucose, pH 6.5;Talbot et al., 1993] containing

2 mM PMSF, for 16 h with gentle shaking, at 28C

4 A small amount of the fungal biomass harvested by filtration throughMiracloth, is placed in a microfuge tube and resuspended in 200ml offixation reagent (2.5% glutaraldehyde in 0.1 M phosphate buffer, v/v,

pH 7.2) or sufficient to cover the mycelial sample Initially the fixation

is carried out under vacuum for 15 min at room temperature andsubsequently at 4C overnight

Figure 20.3 Ultrastructural analysis of autophagy-related membrane compartments Wild-type or atg8D mycelia grown in complete medium for 2 days and subjected to nitrogen starvation for 16 h (in the presence of 2 mM PMSF) were processed for thin- section transmission electron microscopy Numerous autophagic bodies can be detected

in the vacuole of the wild-type strain Scale bar ¼ 0.5 mm.

5 Fixed mycelia are washed 3 times (10 min each) with 0.1 M phosphatebuffer, pH 7.2.

6 The washed mycelial samples are postfixed for 3 h in 250ml of osmiumtetraoxide (1%, w/v)

7 Fixed mycelia are again washed 3 times (10 min each) in 0.1 M phate buffer, pH 7.2

phos-8 Samples are dehydrated in a graded ethanol series (25%, 50%, 75%,100%; 10 min in 500ml each)

9 The samples are then washed twice, for 15 min each, in 250ml ofpropylene oxide

10 Samples are infiltrated in 500ml of propylene oxide-Spurr’s resin (1:1)for 2 h, and then infiltrated overnight in 100% Spurr’s resin

11 Next, samples are embedded in Spurr’s resin and polymerized overnight

at 70C in EMS embedding molds (Electron Microscopy Sciences,USA)

12 Ultrathin (80 nm) sections are generated using Leica Ultracut UCT andmounted on 200 mesh copper grids

13 Mounted sections are stained for 15 min at room temperature with amixture of 2% uranyl acetate and 2% Reynolds’ lead citrate (10mlfor each grid) and examined using a JEM1230 transmission electronmicroscope ( Jeol, Tokyo, Japan) at 120 kV

2.4 Monodansylcadaverine (MDC) staining of

autophagic vesicles

MDC is an acidotropic dye that labels late stage autophagosomes or hagic vesicles (Niemann et al., 2001) MDC staining was successfully used formonitoring the increased autophagic activity in nitrogen-starved Magnaporthemycelia (see Fig 20.2), and the incorporation of MDC into late-stageautophagosomes or autophagic vesicles was inhibited by pretreatment with

autop-WM, the chemical inhibitor of autophagic sequestration Furthermore,MDC staining with the conidiating cultures of Magnaporthe at different stageslikely reflects the natural induction of autophagy (either basal levels ordevelopmentally-induced), without starvation MDC-incorporated compart-ments were copious in the conidiation-specific cell types, including aerialhyphae, and both young and mature conidia An important limitation is thatMDC staining fails to differentiate between late stage autophagosomes andautophagic (acidified) vacuoles

Procedure

1 Magnaporthe wild-type or atg8D strains are cultured on Prune agarmedium (PA; per liter: prune juice 40 ml, lactose 5 g, yeast extract 1 g,agar 20 g) in the dark at 28C for 2 days

2 The wild-type and atg8D strains are then subjected to constant tion (using overhead room lighting) to induce conidiation at roomtemperature.

illumina-3 At 6, 12, and 48 h after photoinduction, the conidiating cultures of thewild-type and atg8D strain are harvested by scraping with an inoculationloop and stained with 0.05 mM MDC solution (stock solution: 5 mM innormal phosphate buffered saline, pH 7.0) at 37C for 15 min TheMDC is then washed out with PBS before microscopic observation

4 MDC-stained mycelia are observed using an epifluorescence microscopeequipped with the following filter sets: excitation wavelength 350 nm,emission 320 to 520 nm

2.5 LysoTracker-based visualization of vacuoles and

vesicular compartments

LysoTracker Green DND-26 and LysoTracker Red DND-99 Molecular Probes, USA) are commonly used to stain and visualize acidiccompartments, including autophagic compartments (Liu et al., 2005; Scott

(Invitrogen-et al., 2004) LysoTracker dyes differ slightly from MDC and label acidifiedautophagic vacuoles (Fig 20.4) but fail to incorporate into late-stageautophagosomes In conidiating aerial hyphae of Magnaporthe, MDC stain-ing was prominent, while very rare staining of LysoTracker Green DND-26

or LysoTracker Red DND-99 was detected Both the LysoTracker- andMDC-labeled spherical compartments were evident in conidia, indicatingthat aerial hyphae are devoid of vacuoles that are mostly formed in conidia.One major drawback of the use of LysoTrackers is the inability to performco-localization studies with RFP-Atg8 labeled vesicles (Deng and Naqvi,unpublished data)

Figure 20.4 Mycelia from the wild-type B157 strain of Magnaporthe was stained with LysoTracker Green DND-26 or LysoTracker Red DND99 and subjected to the requisite epifluorescence microscopy to visualize acidified autophagic vacuoles Bar ¼ 5 mm Different morphological variants of the fungal vacuoles (numerous small vesicles, top,

or big round vesicles, bottom) are detected through LysoTracker DND staining.

1 Magnaporthe wild-type and atg8D strains are grown on PA medium in thedark at 28C in an incubator for 2–3 days The diameter of the resultingfungal colonies is about 1–2 cm at this stage

2 Conidiation is induced in the wild-type and atg8D colonies by subjectingthem to constant illumination at room temperature

3 At 6, 12, and 48 h after photoinduction, the wild-type and atg8D idiating cultures are harvested by scraping with an inoculation loop insterile distilled water and collected through filtration using Miracloth.The harvested biomass is then stained with 50 nM LysoTracker GreenDND26 solution (Invitrogen-Molecular Probes, USA; the stock is 1 M

con-in DMSO) for 10 mcon-in at 37C Staining with LysoTracker Red DND99(1 M in DMSO; Invitrogen-Molecular Probes, USA) followed the sameprotocol as described previously

4 The stained mycelia are observed with epifluorescence microscopy usingthe following filter sets: excitation wavelength 488 nm, emission 505 to

530 nm LysoTracker Red DND99 staining is visualized using 543 nmexcitation and a 560 nm emission filter

2.6 Analysis of glycogen sequestration and estimation

of glycogen content

In most eukaryotic cells, glycogen is stored as a carbohydrate reserve.Autophagy is involved in glycogen catabolism, which occurs in response

to depletion of nutrients or to particular growth/differentiation conditions

In S cerevisiae, glycogen can be degraded in the cytoplasm or inside vacuolarcompartments In the cytoplasm, Gph1 mediates glycogen breakdownresulting in the release of glucose 1-phosphate (G1P) (Hwang et al.,

1989) In vacuoles, glycogen degradation is catalyzed by the vacuolarglucoamylase (Colonna and Magee, 1978), which produces glucose6-phosphate (G6P) The vacuolar degradation of glycogen is sporulation-specific and probably relies on autophagy for sequestration and delivery ofglycogen (Fonzi et al., 1979; Francois and Parrou, 2001; Wang et al., 2001;Yamashita and Fukui, 1985)

To monitor the sequestration of glycogen in budding yeast, KI-I2

staining or iodine vapor exposure is widely used (Hwang et al., 1989;Wang et al., 2001) In M oryzae, detection of glycogen by KI-I2staining

in differentiating conidia (Park et al., 2004; Thines et al., 2000) or by PAS(Periodic acid-Schiff ) staining with sectioned fungal materials (Clergeot

et al., 2001) has been reported Total glycogen content in the yeast or fungalcells can be determined enzymatically by hydrolyzing the extracted glyco-gen witha-amylase and amyloglucosidase, and then measuring the releasedglucose in a colorimetric assay containing glucose oxidase, peroxidase ando-dianisidine (Gascon and Lampen, 1968; Lillie and Pringle, 1980) The kits

for total starch (Megazyme, UK) estimations are also commercially availableand have been successfully used in Magnaporthe samples (Deng and Naqvi,unpublished data).

Procedures

Iodine vapor staining:

1 The fungal strain of interest is subcultured on PA medium andallowed to grow in the dark at 28C for 2 days

2 The dark-grown cultures are then subjected to constant illumination

at room temperature to induce conidiation

3 At 0, 2, and 4 d after photoinduction, the culture dishes containingthe colonies are inverted directly (with the lids removed) over a glassbeaker containing iodine crystals for approximately 15 min Caution:This step has to be carried out in a proper fume hood using appropri-ate safety measures

4 The iodine-stained colonies (Fig 20.5) are photographed immediately.Estimation of total glycogen in fungal tissue(s)

1 The fungal strains (wild type or the autophagy-deficient mutant ofinterest) are grown on prune agar medium in the dark at 28C for 2 days

2 Conidiation is induced in the fungal strains by subjecting them toconstant illumination at room temperature

3 At 0, 2, and 4 d after photoinduction, colonies from the respectivestrains are harvested by scraping the colony surface with inoculationloops in approximately 10–15 ml of sterile distilled water and thenfiltered using sterile Miracloth (Calbiochem, USA)

4 The harvested biomass is ground to a powder in liquid nitrogen using

a mortar and pestle

5 The total glycogen content is estimated using the Megazyme Total StarchKit (Megazyme, UK) as instructed Total glycogen content is normalized

to the wet weight of the fungal biomass used for the estimation

Figure 20.5 Analysis of glycogen accumulation during Magnaporthe conidiation.

A wild-type strain grown in the dark for 2 days was subjected to constant illumination for the specified time intervals, and finally exposed to iodine vapor for 15 min and quickly photographed.

2.7 Comparative proteomics for identifying the targets

of autophagic degradation

Because autophagy is a catabolic process responsible for lysosomal (vacuolar)degradation of proteins, a block of the autophagy pathway will lead toaccumulation of proteins that are destined for autophagic degradation Toidentify proteins that are regulated by autophagic degradation during con-idiogenesis, we performed an SDS-PAGE fractionation of total lysates from4-day-old conidiating cultures of the wild-type, the atg8D mutant, and thecomplemented strains Mass spectrometry was then used to identify theproteins that showed differential accumulation A vacuolar a-mannosidase(MGG_04464) in the Magnaporthe genome was present in the atg8D mutantbut absent in the wild-type or the complemented strains In yeast, Ams1 isdelivered to the vacuoles via Cvt and autophagy pathway and acts as avacuolar hydrolase for free oligosaccharide degradation (Chantret et al.,2003; Hutchins and Klionsky, 2001) Another protein identified in thisassay is Gph1 (MGG_01819), which is responsible for cytoplasmic glycogendegradation (Hwang et al., 1989) The third protein that accumulates inatg8D mutant but not the wild type was a methionine synthase(MGG_06712) In S cerevisiae, Pichia pastoris, Cryptococcus neoformans, and

C albicans, the cobalamin-independent methionine synthase is required forthe growth of yeast or mycelial cells on methionine-free minimal media(Burt et al., 1999; Csaikl and Csaikl, 1986; Huang et al., 2005; Pascon et al.,

2004) In the human pathogenic fungi C neoformans and C albicans,methionine synthase acts as a virulence factor (Burt et al., 1999; Pascon

et al., 2004) An aconitase (MGG_03521) function showed reduced sion on loss of autophagy All the identified proteins are involved in fungalmetabolism and potentially subjected to regulation by autophagy, thusvalidating the importance of such a comparative proteomics approach inidentifying the potential targets of the autophagic degradation pathway forproteins

4 The harvested fungal biomass is ground to a fine powder in liquidnitrogen, using an autoclaved mortar and pestle, and resuspended in

300 ml of extraction buffer (10 mM Na2HPO4, pH7.0, 0.5% SDS,

1 mM DTT and 1 mM EDTA) Lysates were cleared by centrifugation

at 12,000 g for 30 min at 4C Protein concentration in the supernatantfraction is determined by Nanodrop ND-1000 spectrophotometer(Thermo Scientific, USA).

5 Normalized protein sample from each extract is fractionated by PAGE (10%), mixing with equal volume of loading buffer (Bio-Rad,USA)

SDS-6 Protein bands of interest (showing differential expression levels betweenthe wild-type and atg mutant) are excised from the gel

7 In-gel digestion and peptide extraction are performed using the followingprotocol: http://www.millipore.com/userguides.nsf/dda0cb48c91c0fb6852567430063b5d6/2265a5645f93475785256cbe00558a60/$FILE/P36505D.pdf

8 Proteins of interest are identified by peptide sequencing or TOF mass spectrometry

MALDI-3 Concluding Remarks

A recent review (Klionsky et al., 2007) discusses methodologies to studythe dynamics of autophagy in yeast and mammalian cells The methodsdescribed previously measure the steady-state levels of autophagy, coveringthe induction, vesicle formation, and vesicle fusion Very few assays thatmonitor the complete autophagic flux have been reported in filamentousfungi One such assay relates to measuring the total protein breakdown instarved cells in wild-type versus an autophagy-deficient mutant in yeast(Tsukada and Ohsumi, 1993) An alternate method requires quantification

of the total alkaline phosphatase activity in a yeast strain expressing a truncatedform of Pho8, Pho8D60, which exists as an inactive precursor in the cytosoland is entirely dependent on autophagy to be delivered to the vacuole for itsactivity during starvation Pho8D60 gains its phosphatase activity on specificprocessing by vacuolar enzymes so that the measurement of its activity can be

an indicator of the fusion of autophagosomes with the vacuole and thesubsequent processing therein (Kirisako et al., 1999; Wang et al., 2001) Seethe chapters by Noda and Klionsky, Cabrera and Ungermann, and Mayer inthis volume for information on the Pho8D60 assay

ACKNOWLEDGMENTS

We thank Yang Ming for generating the atg1 deletion mutant, and the Fungal Patho-biology group for discussions and suggestions We are grateful to S Naqvi for critical reading of the manuscript Intramural research support from the Temasek Life Sciences Laboratory, Singapore is gratefully acknowledged.

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