cell growth is tightly linked with the cell 's perception of its nutritional environment . in particular , microorganisms , such as the yeast saccharomyces cerevisiae , that spend most of their time in the stationary phase in the wild ,
therefore , yeast is a good model organism for the study of nutrient detection and response .
autophagy is one of several responses to nutrient starvation ( klionsky and ohsumi 1999 ) .
a large number of cytoplasmic components are nonselectively enclosed within a double - membrane structure called an autophagosome , in which they are transported into the vacuole / lysosome to be degraded by resident hydrolases
. such turnover of a large amount of cytoplasm mediated by autophagy is essential for survival under nutrient - depleted conditions .
we have isolated several genes essential for autophagy ( termed apg ) and have been investigating the function of the gene products ( tsukada and ohsumi 1993 ; funakoshi et al . 1997 ; matsuura et al .
1998 ) . genetic and morphological analyses revealed that the degradative process of autophagy shares mechanistic components with the cytoplasm - to - vacuole targeting ( cvt ) pathway ( harding et al .
; baba et al . 1997 ) , which is biosynthetic , delivering a resident hydrolase , aminopeptidase i ( api ) , to the vacuole ( klionsky and ohsumi 1999 ) .
on the other hand , autophagy and the cvt pathway are distinct in many aspects .
the two pathways appear to be regulated separately ; the cvt pathway is mainly observed under growing conditions , whereas autophagy is induced by starvation ( baba et al .
furthermore , cvt vesicles and autophagosomes , the vesicles formed in the cvt pathway and autophagy , respectively , are clearly different in size ( baba et al . 1997 ) .
recently , the t - snare tlg2 and sec1-homologue vps45 were found to be required for the cvt pathway , but dispensable for autophagy , suggesting that these two pathways are mechanistically distinct ( abeliovich et al . 1999 ) .
tor is a phosphatidylinositol kinase - related kinase that promotes cell cycle progression in response to nutrient availability ( thomas and hall 1997 ) .
treatment with the immunosuppressant rapamycin , a specific inhibitor of tor , induced cell cycle arrest at g0 .
for example , accumulation of glycogen , repression of genes that are stimulated in growing cells , and stimulation of genes that are induced during starvation , all result from inhibition of tor ( hardwick et al .
our laboratory has found that rapamycin induces autophagy in yeast ( noda and ohsumi 1998 ) .
however , the molecular mechanism by which tor negatively regulates autophagy remains to be determined .
here , we present evidence that apg1 , a protein kinase essential for autophagy , plays a pivotal role in induction of tor - regulated autophagy .
an nh2-terminally truncated ( 8 amino acid ) form of apg1 open reading frame , subcloned into pgbd - c2 vector , was used as bait to screen a yeast genomic library , and interacting proteins were identified by dna sequencing . a dna fragment including the entire apg17 gene was cloned from yeast genomic dna using pcr .
the kinase - negative apg1 mutation was obtained using the quikchange site - directed mutagenesis kit ( stratagene ) .
antibody against apg13 protein was raised against a glutathione s  transferase ( gst)-apg13 fusion protein .
specifically , a 1.9-kb bglii - xbai fragment of the apg13 gene coding for 396 amino acids of apg13p was subcloned into the vector pgex-2 t .
the expressed fusion protein was purified with glutathione - sepharose 4b ( amersham pharmacia biotech ) .
expression and purification of the fusion protein was carried out according to the manufacturer 's instructions .
immunoprecipitation , kinase assay , and immunodetection of nh2-terminally hemagglutinin ( ha)-tagged apg1 ( apg1 ) were performed as described previously ( kamada et al .
apg1 was immunoprecipitated with anti - ha mab ( 16b12 ; babco ) , and an in vitro protein kinase assay was performed in the presence of [p]atp and myelin basic protein ( mbp , substrate ) .
yeast cells exponentially grown in yepd medium were treated with zymolyase 100 t ( seikagaku kogyo ) to generate spheroplasts .
the resultant spheroplasts were treated with or without 0.2 g / ml of rapamycin and broken by resuspending in lysis buffer ( pbs , ph 7.4 , 1 mm edta , 1 mm egta , 2 mm na3vo4 , 50 mm kf , 15 mm na2h2p2o7 , 15 mm p - nitrophenylphosphate , 20 g / ml leupeptin , 20 g / ml benzamidine , 10 g / ml pepstatin a , 40 g / ml aprotinin , 1 mm pmsf , and 0.5% tween-20 ) .
cell lysate was cleared by 10-min centrifugation at 6,500 g and 30-min incubation with protein g 
apg1 in the cleared cell lysate was bound to anti - ha mab , and apg13 was detected with anti - apg13 antibody .
the resultant immunoprecipitates were also analyzed by protein kinase assay and immunoblot with anti - ha . for in vivo labeling of apg13 , cells ( tfd13-w3 ) expressing apg13 were in vivo - labeled with 50 ci of s ( trans , icn ) for 10 min , or 50 ci of pi overnight in sd medium , and transferred to yepd or nitrogen - depleted medium sd(n ) for 1 h. apg13 protein was immunoprecipitated following tca precipitation .
immunoprecipitated apg13 was treated with 5 u of alkaline phosphatase for 1 h at 30c .
progression of autophagy was estimated by the increase of alkaline phosphatase activity in the cells expressing a cytosolic proform of the phosphatase protein ( pho860p ; noda et al .
in an effort to study the mechanism of autophagy induction , we focused on the apg1 gene , which encodes a protein kinase whose activity is essential for autophagy ( matsuura et al .
nh2-terminally ha - tagged apg1 ( apg1 ) was immunoprecipitated with anti - ha ascite and the resultant immunocomplex was analyzed using an in vitro kinase assay .
apg1 kinase activity was found to be highly elevated in cells grown under starvation conditions ( fig .
1 a ) . after a 6-h incubation in nitrogen - depleted medium , sd(n )
, the amount of activated apg1 had apparently increased , and was accompanied by slower gel migration , presumably because of autophosphorylation ( fig .
the increase in apg1 kinase activity is not due to this apparent increase in the protein amount , because shorter treatments with rapamycin ( for example , see fig .
apg1 activity was also increased by rapamycin treatment , but the effect of rapamycin was abolished in a rapamycin resistant tor1 mutant ( tor1 - 1 ; kunz et al .
these results suggest that apg1 activation is required for the induction of autophagy and that it is mediated by tor proteins . a kinase - negative apg1 mutant ( k54a ;
this indicates not only that the enhanced apg1 kinase activity is required for autophagy , but that basal apg1 activity in growing cells ( fig .
next , we performed a two - hybrid screening with apg1 as bait to identify apg1-associating proteins , which may regulate apg1 activity .
the following three genes were obtained from the screen : apg13 ( funakoshi et al .
1997 ) and two novel genes , which were subsequently found to be essential for either autophagy or the cvt pathway , or both .
one gene , designated as apg17 ( ylr423c ) , was essential for only autophagy and was not required for the cvt pathway ( fig .
2 a ) . the other , cvt9 ( harding et al . 1996 ; d.j . klionsky , personal communication ) , was required for the cvt pathway , but not for autophagy . among the 16 apg genes discovered so far ,
it is interesting to note that apg1 binds to proteins whose function is specific to either autophagy ( apg17 ) or the cvt pathway ( cvt9 ) .
overexpression of apg1 in an apg13 mutant partially rescues the autophagy defect ( funakoshi et al .
similarly , the apg17 mutant was also rescued by overexpression of apg1 ( data not shown ) , indicating that these three genes interact functionally .
in the apg13 mutant , attenuated apg1 activity was observed . in rapamycin - treated apg17 cells
, apg1 activity also was found to be largely impaired ( 20% of the wild - type ) . on the other hand ,
deletion of cvt9 , which is not needed for autophagy , resulted in rapamycin - induced activation of apg1 to 50% of wild - type .
the effects of deleting apg13 and apg17 on apg1 activity are not the result of a general autophagy defect , because deletion of other apg genes , such as apg5 ( mizushima et al .
1998 ) , does not affect the activation of apg1 ( data not shown ) .
these results indicate that the activated state of apg1 is required for autophagy induction , and that apg13 and apg17 play a key role in the activation of apg1 in response to tor inhibition .
the next question we addressed was how these apg1-associating proteins transmit the starvation signal from tor to apg1 .
overexpression of apg13 resulted in a smeared apg13 band on the immunoblot caused by retarded migration , indicating that it was modified in some way ( fig .
this modified form was observed only in growing cells , and after starvation or rapamycin treatment , the slower - migrating form disappeared . in particular , it was noted that the disappearance of the slower - migrating form occurred within five minutes after rapamycin treatment . in vivo labeling and in vitro phosphatase treatment revealed that the apg13 bandshift was due to phosphorylation ( fig .
these results suggest that apg13 is phosphorylated in a tor - dependent manner , which was confirmed by the observation that dephosphorylation of apg13 in response to rapamycin was not seen in tor1 - 1 cells ( fig .
the dephosphorylated , faster - migrating form of apg13 in starved cells was rapidly phosphorylated after readdition of yepd medium ( fig .
3 d ) , suggesting that the phosphorylation state of apg13 is extremely sensitive to nutrient conditions .
this excludes the possibility that the phosphorylated form of apg13 is degraded upon starvation and apg13 is de novo synthesized after medium addition .
the faster - migrating form of apg13 ( as well as the slower - migrating form ) was found to be labeled with pi ( fig
. 3 b , lane 8) , suggesting that apg13 remains partially phosphorylated under starvation conditions . to confirm that there is a physical association between apg1 and apg13
a vacuolar protease - deficient strain was used for these experiments because apg13 is quite labile in cell lysate . when apg1 and apg13 were expressed from a high - copy plasmid , only the faster - migrating form of apg13
3 e ) , indicating that the hyperphosphorylated form of apg13 has little affinity for apg1 .
next , we performed the experiment using a low - copy plasmid to more closely approximate physiological cellular conditions .
the amount of apg13 bound to apg1 increased rapidly ( as quickly as 10 min ) after rapamycin treatment ( fig . 3 f ) , which corresponds well to the time course of apg13 dephosphorylation .
these results strongly indicate that tor negatively regulates apg1 kinase activity by means of ( hyper)phosphorylation of apg13 , which reduces the affinity of apg13 for apg1 . from these results
, we hypothesized that apg13 binding to apg1 is required for the induction of autophagy , but not for the cvt pathway .
the two - hybrid assay was used to determine the apg1-binding site on apg13 , using various apg13 open reading frame fragments as prey . a central 89-amino acid region ( 432520 )
we also isolated a cooh - terminal truncated form of apg13 , apg13(1448 ) , whose ser449 was mutated to a stop codon using in vitro mutagenesis ( kaiser et al .
this mutant has a mutation within the putative apg1-binding site , and is unable to associate with apg1 , as confirmed by the two - hybrid assay and coimmunoprecipitation ( fig .
4 a , and data not shown ) . this result indicates a role for a domain of apg13 around amino acid 448 in binding apg1 .
we tested several cooh - terminal truncated apg13 mutants including apg13(1448 ) for autophagy activity .
4 b , bottom ) , presumably due to the absence of the apg1-binding domain .
another cooh - terminal truncated form , apg13 ( 1568 ) , which contains the entire putative apg1-binding domain , displayed partial , but significant , autophagic activity , when compared with apg13(1448 ) .
apg13(1568 ) was less competent for autophagic import than full - length apg13 , suggesting that additional sequences downstream of the apg1 binding site are important for maximal activity .
on the other hand , apg13(1448 ) partially , but significantly , rescued vacuolar - targeting of precursor api ( fig .
4 b , top ) , suggesting that this truncated protein is still functional for the cvt pathway .
apg1 activity in the apg13 mutant was partially restored in the presence of either apg13(1448 ) or apg13(1568 ) , and was fully recovered in the presence of the whole apg13 construct ( fig . 4 c ) .
the kinase activity of the apg13(1568 ) transformant was clearly higher than that of the apg13(1448 ) transformant .
these results confirmed the hypothesis that the apg1apg13 association and subsequent activation of apg1 are required for autophagy induction in response to starvation .
it is currently thought that tor signaling in yeast is bifurcated ( thomas and hall 1997 ) .
one pathway involves the small gtpase rho1 , which is responsible for actin organization and is not affected by rapamycin .
the other involves tap42 , a rapamycin - sensitive phosphatase - associating protein that is necessary for the initiation of protein translation and amino acid permease turnover ( di como and arndt 1996 ) . tap42
is known to be located directly downstream of tor , and is required for tor - mediated signaling , especially the rapamycin - sensitive branch ( jiang and broach 1999 ) . to investigate this issue further , we tested the ability of a tap42 mutant ( tap42 - 11 ; di como and arndt 1996 ) to induce autophagy at a nonpermissive temperature .
accumulation of vacuolar autophagic bodies in tap42 cells was found to be comparable to that in wild - type cells , confirming that induction of autophagy is not controlled by tap42 ( fig . 5 ) .
furthermore , deletion of npr1 , whose product is a protein kinase negatively regulated by tap42 ( schmidt et al .
1998 ) , did not affect the induction of autophagy ( data not shown ) .
therefore , we concluded that tap42 does not transmit a signal from tor in the autophagy induction pathway .
the association between apg1 and apg13 is negatively regulated by tor signaling . in nutrient - rich conditions ,
dephosphorylated apg13 possesses a high affinity for apg1 , which is activated upon apg13 binding , leading to induction of autophagy .
this tight apg1apg13 association is required for autophagy , but not for the cvt pathway , an observation that supports a model in which the apg1apg13 complex plays an important role in switching from the cvt pathway to autophagy in response to nutrient conditions ( fig .
the function of apg17 is still unclear , but our preliminary results suggest that it may be involved in the apg1apg13 interaction . when both apg1 and apg13 were overexpressed , apg1apg13 binding was observed , even in cells grown in yepd ( fig .
3 d ) , resulting in a small amount of apg1 activation , insufficient to induce autophagy ( e.g. , see fig
protein sorting to the plasma membrane and vacuolar degradation of amino acid permeases , gap1 and tat2 , are also regulated in response to nutrient conditions , and are dependent upon tor , npr1 , and tap42 ( but not on apg1 ; roberg et al .
tap42 plays a key role in the rapamycin - sensitive tor pathway , suppressing npr1 activity ( schmidt et al .
our observation that tor regulation of apg1 activity and autophagy induction is rapamycin - sensitive , but tap42-independent , implies that the apg1apg13 interplay comprises a novel tor signaling pathway regulating autophagy induction .
it is possible that tor directly phosphorylates apg13 , but this remains to be investigated .
recently , nuclear transport of transcription factor gln3 has been shown to be under the control of tor signaling , but the involvement of tap42 in this system is still controversial ( beck and hall 1999 ; cardenas et al . 1999 ) .
deletion of gln3 did not affected autophagy ( data not shown ) . in mammalian cells , two targets of mammalian tor ( mtor )
have been identified : 4e - bp1 and p70s6k ( thomas and hall 1997 ) .
another recent study reported a relationship between mammalian tor and autophagy ( shigemitsu et al .
apg1 homologues of caenorhabditis elegans ( ogura et al . 1994 ) and mammals ( yan et al .
 autophagy is a membrane trafficking to vacuole / lysosome induced by nutrient starvation . in saccharomyces 
 cerevisiae , tor protein , a phosphatidylinositol kinase - related kinase , is involved in the repression of autophagy induction by a largely unknown mechanism . here , 
 we show that the protein kinase activity of apg1 is enhanced by starvation or rapamycin treatment . 
 in addition , we have also found that apg13 , which binds to and activates apg1 , is hyperphosphorylated in a tor - dependent manner , reducing its affinity to apg1 . 
 this apg1apg13 association is required for autophagy , but not for the cytoplasm - to - vacuole targeting ( cvt ) pathway , another vesicular transport mechanism in which factors essential for autophagy ( apg proteins ) are also employed under vegetative growth conditions . 
 finally , other apg1-associating proteins , such as apg17 and cvt9 , are shown to function specifically in autophagy or the cvt pathway , respectively , suggesting that the apg1 complex plays an important role in switching between two distinct vesicular transport systems in a nutrient - dependent manner . 