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Isolation and characterization of AAP1. A gene encoding an alanine/arginine

aminopeptidase in yeast

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THEJ O U R N A L OF BIOLOGICALCHEMISTRY Val. 268, No. 19, Issue of July 5 , pp. 14310-14315,1993
0 1993 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

Isolation and Characterizationof AAPI

A GENEENCODINGANALANINE/ARGININEAMINOPEPTIDASEINYEAST*

(Received for publication, December 1, 1992, and in revised form, February 3, 1993)

Daniel R. Caprioglio, Christopher Padilla, and Margaret Werner-WashburneS

From the Biology Department, University of New Mexico, Albuquerque, New Mexico 87131

The yeast A A P l gene, encoding a putative amino- onine aminopeptidase, which is responsible for cleaving the
peptidase, was isolated based on its ability to suppress NH2-terminal methioninefrom many newly synthesized pro-
the temperature-sensitive growth on nonfermentable teins in both eucaryotic and procaryotic cells. In Escherichia
carbon sources of sprb, a stationary phase regulatory coli and Salmonella typhimurium, methionine aminopeptidase
mutant. A A P l was physically mapped to chromosome (MAP)' genes are essential for growth (8).In yeast, the MAPl
VI11 between PUT2 and C U P l . Sequence analysis of gene encoding a methionine aminopeptidase has been cloned
the A A P l gene showed a 1581-nucleotide open reading and sequenced (9). The MAPl gene is not essential for yeast
frame capable of encoding a 59-kilodalton protein. The
viability, but disruptionof MAPl results ina decrease in both
protein encoded by this open reading frame exhibits
approximately 40%sequence identity to human, rat, growth rate and colony size (9).
andmouse aminopeptidases. In limited regions, se- In this work, we describe the cloning, sequencing, deletion,
quence identity between Aapl and the mammalian and overexpression of the AAPl gene, which suppresses the
aminopeptidases ranges from 53%to 93%. Insertional temperature-sensitive growth onnon-fermentablecarbon
inactivation of the A A P l gene resulted in a decrease sources of a stationary-phase regulatory mutant (spr5). We
in glycogen accumulation and the loss of the major present evidence that the AAPl gene, which exhibits exten-
bandof argininetalanine aminopeptidase activity. sive sequence identity with mammalian zinc aminopeptidases,
Strains carryingthe A A P l gene on ahigh copy plasmid encodes anonessential, alanine/arginine aminopeptidase that
show an increase in the major argininefalanine ami- acts asa positive effector of glycogen accumulation.
nopeptidase activity, a dramatic increase in glycogen
accumulation, and an increase in transcription from a MATERIALS ANDMETHODS
vector carrying lacZ fused to the promoter of a gene
Strains-S. cerevisiae strains used in this study are as follows:
(SSA3) expressed during post-diauxic and stationary DSlO(MATa his3-11, 15 urd-52 Atrpl leu2-3, 112 lys2) (lo), DS13
phases of the culture cycle. We conclude that although (MATa his3-11,15urd-52 Atrpl leu2-3,112) ( I l ) , MW443 (MATal
the A A P l gene is not essential for viability, the Aapl MATa his3-11, 15/his3-11, 15 ura3-52/urd-52 AtrpllAtrpl leu2-3,
protein positivelyaffects glycogen accumulation in I12/leu2-3,112) ( l l ) , DC27 (MATa his3-11, 15 urd-52 Atrpl leu2-
yeast. 3, 112 lys2 spr5-27), DC106 (MATa his3-11, 15 urd-52 Atrpl leu2-
3, 112 lys2 aapl::HIS3), DC126 (MATa his3-11, 15 urd-52 Atrpl
leu2-3, 112 lys2 aapl::LEU2), DC20 (MATa his3-11, 15 ura3-52
Atrpl leu2-3, 112 lys2, sprl-20) (this study), and JC302-26B (MATa
his4 leu2 urd-52 rasP:LEU2) (12).
A number of distinct proteases have been identified in the Media and Growth Conditions-Media used were YPD (1%yeast
yeast Saccharomyces cerevisiae (1, 2). Among the best studied extract, 2% peptone, 2% glucose), YPGal (1% yeast extract, 2%
arethe vacuolar proteases, which are involved inprotein peptone, 2% galactose), YPGly (1%yeast extract, 2% peptone, 3%
turnover, especially during sporulation and entry into station- glycerol), YPAc(1%yeast extract, 2% peptone, 2% potassium acetate,
pH 5.5), SC (synthetic complete medium lacking the appropriate
ary phase (2); the enzymes involved in the ubiquitin-pathway supplements to maintain selection of specific marker genes), and
of ATP-dependent protein degradation,which is essential for potassium acetate (1%potassium acetate, 0.1% yeast extract, 0.5%
survival during stationary phase (3); and the proteases re- dextrose) (13). Unless otherwise noted, cells were grown at 30 "C.
quired for maturationanddegradation of mating factors: Cloning of the AAPl Gene-To select for genes that complement
Kexlp, Barlp, anddipeptidylaminopeptidase A (1).Fourteen the spr5 phenotype of no growth on nonfermentable carbon sources
distinct aminopeptidase and dipeptidase activities have been such as YPAc and temperature sensitivity on YPGal, which requires
characterized in yeast (4),but their in uiuo roles are unclear. mitochondrial function (14), DC27 (spr5-27) was transformed with
the YEpl3 multicopy-based plasmid library (15, 16). Transformants
In other eucaryotes, aminopeptidaseshave been suggested to estimated to carry 3.5 genome equivalents were tested for reversion
play significant roles in growth and differentiation (5-7). to growth on YPGal at 37 "C. pAAPl was isolated from one of the
One of the best studied aminopeptidases in yeast is methi-transformants. DC27 strains carrying pAAPl were cured of the
plasmid and found to be unable to grow on YPGal at 37 "C in the
* This work was supported by National Science Foundation Grant absence of the plasmid. Retransformation of DC27 with pAAPl
DIR-9101939 (to D. R. C.) and National Science Foundation Grants restored growth on YPGal at 37 "C and on YPAc at all temperatures.
DCB-9000556 and PYI-9000556 (to M. W.-W.). The costs of publi- Physical Mapping-Chromosomal assignment was determined by
cation of this article were defrayed in part by the payment of page hybridization of an AAPl probe to a ChromoDiHybridizer blot of
charges. This article must therefore be hereby marked "advertise- pulsed-field-separated yeast chromosomes, purchased from Clontech
ment" in accordance with 18 U.S.C. Section 1734 solely to indicate (Palo Alto, CA). An ordered collection of yeast genomic DNA cloned
this fact.
The nucleotide sequence(s) reported in this paper has been submitted The abbreviations used are: MAP, methionine aminopeptidase;
to the GenBankTM/EMBLData Bankwith accession numberfs) YPD, yeast extract/peptone/dextrose medium; YPAc, yeast extract/
L12542. peptone/acetate medium; YPGal, yeast extract/peptone/galactose
$.Towhom correspondence should be addressed. Tel.: 505-277- medium; YPGIy, yeast extract/peptone/glycerol medium; aa, amino
9338: Fax: 505-277-0304. acid(s); kb, kilobase pair(s).

14310
 Isolation and Characterizationof the YeastAAPl Gene
into bacteriophage X was generously provided by L. Riles and M.
Olson (Washington University, St. Louis, MO).
Plasmid Construction and GeneProbes-All recombinant DNA
methods were performed using standard procedures (17). A 4.4-kb
EcoRI-KpnI fragment of pAAPl (containing the AAPlgene) (Fig. 2)
was subcloned into EcoRIIKpnI-cut pUC19 (18), yielding pDC29.
The 4.4-kb EcoRI-Sal1 fragment of pDC29 was cloned into EcoRI/
SalI-cut pRS413, a centromericvector carrying theHIS3 marker (19),
to yield pDC3O. The plasmid pDC31 was made by cloning the 1.7-kb
BglII-EcoRI fragment of pDC29 intoBamHIIEcoRI-cut pRS413.
pDC32 was constructed by cloning the 2.6-kb BglII-BarnHI fragment
of pDC29 into BamHI-cut pRS413. pDC33 was made by cloning the
3.4-kb PstI fragment of pDC29 into PstI-cut pRS415, a centromeric
vector carrying the LEU2 marker (19). pDC38 was constructed by
cloning the 2.8-kb EcoRI-XbaI restriction fragment from pDC29 into
the EcoRIIXbaI sites of pRS413.
AAPl Gene Disruption-To disrupt the AAPl gene, the 1.8-kb
XbaI fragment (between the internal XbaI site and the XbaI site in
pUC19) was deleted from pDC29 to yield pDC34. pDC34 was then
14311
YPGal at 37 "C and on YPAc at 30 and 37 "C (Fig. 1). In
addition to suppression of these phenotypes, the presence of
pAAPl inDC27 (spr5-27)or control (DSlO)cells is associated
with more rapid growth on acetate-based medium (Fig. I),
hyperaccumulation of glycogen (Fig. 6), and increased expres-
sion of the HSP70-related SSA3 gene during entry into sta-
tionary phase (Fig. 7).
To determine whether the AAPl gene present in low copy
number could also suppress the spr5-27 mutant phenotype,
pDC3O was constructed, containing the 4.4-kb EcoRI-KpnI
fragment of pAAPl (Fig. 2). spr5 mutant cells carrying this
construct exhibitweak suppression of the mutant phenotype
(Fig. 1).
Strain:
Control 7

I
digested with BglII. A 1.8-kb BamHI fragment isolated from pJJ282
(20) containing the HIS3 gene was cloned into the BglII site of spr5-27
pDC34. The resulting plasmids, pDC35 and pDC36, containing HIS3
in opposite orientations, were digested with EcoRI and XbaI and the spr5-27
resulting fragments used to transform MW443 (Fig. 2). To construct pAAPl
the aapl::LEU2 disruption, pAAPl was digested with EcoRI. The 7.8-
spr5-27
kb fragment containing the EcoRI-KpnI fragment and pBR322 se- pDC3O
quence from 375 through 4363 was isolated and recircularized by
Temperature (OC): 30 37 30 37 30
ligation, producing pDC28. The HindIII-BamHI LEU2-containing
fragment from pJJ283 (20) was cloned into HindIIIIBglII-digested Medium: YPDextrose YPAcetate YPD'12
pDC28, producing pDC37. A 2.8-kb PuuIIIScaI fragment of pDC37
was isolated for transformation into DSlO (Fig. 2). FIG. 1. Suppression of t h e spr5-27 mutant phenotypes of
Northern Analysis-RNA was extracted as previously described poor g r o w t h o n Y P Dat 37 "C and inability to grow on YPAc
(21), 20 pg of total RNA was loaded per lane and separated on a 1% at 30 "C a n d 37 "C, b y AAPI carried on 2 pm (pAAP1) and
agarose-formaldehyde gel (22). Equal loading of RNA was confirmed centromeric (pDC30) plasmids. Strains were grown on solid media
by ethidium bromide staining. The RNA was blotted to Genescreen for 3 days at the designated temperature. YPDextrose, YPD; YPA-
Plus (Du Pont-New England Nuclear), cross-linkedby UVirradiation cetate. YPAc.
to themembrane as suggested (Stratagene, Stratalinker), andhybrid-
ized according tothe manufacturer's instructions (DuPont-New A. Restriction Map
England Nuclear). DNA probes were labeled with [32P]dCTPby the AAPl ORF
random primer method (23,24). The4.4-kb EcoRI-KpnI fragment of
-
E H H P 8 X K
AAPl was used as a probe.
DNA Sequencing-Sequencing of both strands of the AAPI gene
pAAP1 i I I I I I-
-I
5009~
was carried out using oligonucleotide primers and dideoxy chain B. Complementation
termination with Sequenase 2.0 (U. S. Biochemical Corp.). Sequence
analysis was carried out using the TFASTA and BESTFIT programs
of the GCG computer package (25). Analysis of protein sequence was pDC38 -
also carried out with GCG programs (25). poc33
Phenotypic Analysis-To assay for glycogen accumulation, strains poc32
were grown on YPD platesa t 30 "C. Culture plateswere inverted over
iodine crystals in a closed container asdescribed previously (12). The
@-galactosidaseassays were done accordingto Reynolds and Lundblad
(26). Thermotolerance assayswere performed as previously described C. Insertional Inactivations
(11). Briefly, cells were grown a t 24 "C to an Am of0.6-1.0, the E H H P E X
culture was divided in half, and one half was incubated at 39 "C for poc34 I I I I I I
75 min while the other half remained a t 24 "C.
Protein Extraction and Aminopeptidase Assays-Proteins were iso-
lated by published methods (27). Briefly, cells were grown in YPD at
/\
30 "C, collected by centrifugation, and washed once with ice-cold,
distilled H20. Cell lysates in 20 mM Tris (pH 7) were prepared by
homogenizing with a Shake-It-Baby homogenizer (BioSpec Products,
Bartlesville, OK) for 2 min and clarified by a 1-min centrifugation.
Protein quantitation was done using the BCA protein assay (Pierce
Chemical Co.). Proteins were separated by electrophoresisunder
native conditions using a 3.5% acrylamide stacking gel (pH 6.8) and
a 7.5% acrylamide resolving gel (pH 8.8) (28). After electrophoresis,
the aminopeptidase assay was performed as described (27). Briefly,
the gel was incubated for 1 h in 50 mlof 0.1M Tris-HCI, pH 7.5,
containing 50 mM ZnSOl with 25 mg of aminoacyl-NH-naphthylam-
ide (Sigma) at 25 "C. After incubation, the gel was washed several pDC37
times with a 0.2 M potassium phosphate buffer (pH 6.5) and incubated LEU2
in 100 ml of the same buffer containing 31 mg of Fast Garnet GBC FIG.2. Restriction map, complementation, and insertional
(Sigma). This incubation was continued a t 25 "C until color devel- inactivation of AAPI. A, the restriction map of the 4.4-kb EcoRI-
opment was complete. KpnIfragment from pAAP1. Restriction sitesare designated as
follows: E , BglII; E, EcoRI; H, HindIII; K , KpnI; P, PstI; Pu,PuuII;
RESULTS S, ScaI; X, XbaI. B, complementation (+) or lack of complementation
(-) of the spr5-27 temperature sensitivity on YPGal by restriction
The AAPl gene was isolated as a multicopy suppressor of fragments derived from pAAPl and cloned into centromeric vectors.
the spr5-27 mutant phenotype of the inability to grow on C, aapl::HIS3 and aapl::LEU2 disruption mutations.
14312 Isolation
Characterization
and
The AAPl locus was physically mapped to chromosome
VI11 by probing a blot of CHEF-separated yeastchromosomes
(Clontech) with the4.4-kb AAPl probe described under “Ma-
terials and Methods.” The chromosomelocationwas
firmed by probing an orderedX library, constructedby Linda
of the Yeast AAPl Gene
-533 TTT TTTTTCGTAC TCTTGGACTT GGCTGTGGGA GTTTGACACA
- 4 8 0 TTTTTTTATTT TTTTTCCGTT TTTTTCCCTT TTGTTAGCM TCAAACTTTT GACTTTATGA
-420 GTMTCTTAGC TMTCATTTA G M T T A T C T T CAGGGMGCT T A T T C M T T C GTGATTTTTC
-360 TTTGAGGGTTC C T G T T C M G C M G C C A A A T T GCCCTCTCAG C T M T A C C A T G T C C C G T G M
-300 GTACTGCCAAA
-240CTTTTACGTTT
C M C G T T A C C CCATTGCATT ATGACATTAC ACTAGAACCT MTTTCCGTG
GMGGGTCAT TAAIUATTGA CCTGCAGATT M C T G A T C A T TCGACTAAAC
con- -180 TCTGTGCAMT
-120CATTGMGTTA
AAATTACTTA GIMTAGATT TCCATTCGCA CGTATTGAGG GTGTCMTGC
A W C G A M C C A A G C A AAAGTACTCT TGTCTTTCCT MTGGMCTT
- 6 0 TTGAAMCCTA GGACTCCTGC GMGTTAAGM ATTATCTTTT CCGGTATTCT AMTGATCAG

Riles and Maynard Olson (Washington University,St. Louis, ATGGCGGGATTTTACAGGGCTMGTATACGMTAAAGTCACAGGGGAGACGMGTACATG 60

M A G F Y R A K Y T D K V T G E T K Y M 20
MO), with an AAPl probe. Theresults of the X library
CTACTACTCAGATGGM~CACAGATGCTAGMGAGCCTTTCCTTGTTTTGAC~CC~ 120
hybridization analysis indicated that AAPl the gene is located I T T Q M E A T D A K R A F P C F D E P 40
on chromosome VI11 between PUT2 and CUPl. This region MTTTGMGGCMCTTTTGCAGTMCTTT CTCTGMTCGTTTCTGACTCATTTGTCC 180
N L K A T F A V T LS E S F L T H L S 60
does not contain any other mapped yeast genes, suggesting
that AAPl is a newly identified gene. II M C A T G G A T C T C A G G M T G C M T C A M G A G ~ T M ~ TAAC M C T T T T M C A C G
N M D V R N E T I K E -G K -~ K Y ~~
240
~ 80
A complementationanalysis was used to determine the ACTCCMAGATGTCTACGTACTTAG TTThTTGTAGCTGACCTMGATACGTGAGM 300
approximate locationof the AAPl gene on the4.4-kb EcoR1- T P K M S T Y L V F I V A D L R Y V E 100

Kpnl insert of pAAP1 (Fig. 2). Various restriction fragments AGCMTMTTTCCGTATTCCAGTM~GTTTATTCTACCCCAGGG~CG-TTTGGT

S N N F R I P V R V Y S T P G D E K F G
360
120
from the insert were cloned intocentromeric vectors and 420
tested for complementation of spr5-27 temperature sensitiv- 140

ity. None of thesmallerfragments of thepAAPlinsert 480

111 160
complemented the spr5 mutation, suggesting that both the
GTTATTGATTTGTTATTAGCATAGAAkATTCG 540
PstI and Xbal sites lie within AAPl or regions essential for V I D L L L D I E N S 180
its expression (Fig. 2). AGTTTAGATCGCAT CAMGAGTTGCTGAGGTTATTCAGCATGMCTAGCCCACCMTGG 600
Sequence analysis of the 4.4-kb EcoRI-KpnI fragment re- tu S L D R I Q R V A E V I Q H E L A H Q W
~~ ~~~
200

vealed two open reading frames. An open reading frame of TTCGGCMCTTGGTCACCATGMTTGGTGGGMGGCTTATGGTTGAATGMGGTTTTGC

F G N L V T M D W W E G L W L N E G F A
660
220
1581 nucleotides was identified that flanks theBglII site, the 720
region shown to berequired for AAPl complementation (Fig. anuz
240

3). This open reading frame is predictedencode to a polypep- TATGTTACTMCMTTTACMCGTGCTCT~TTTAGATTCATTMGATCTTCCCATCCA

Y V T D N L Q R A L N L D S L R S S H P
780
260
tide of 526 amino acids with an approximate molecular mass 840
A T T G M G T T C C C G T T M T M T G C G G A C G A A A T C M T C ~ T TTTGATGCTATTTCTTAT
of 59 kDa. Analysisof the predicted protein sequence indicates U I E V P V N N A D E I N Q I 2~8 0

that the Aapl protein is relatively hydrophilic, contains no TCTAAAGGATCTTCTTTATTGAGGATGATTTC AMTGGTTGGGTGMGAMCTTTTATT 900
S K G S S L L R M I S K W L G E E T F I 300
long hydrophobic regions, and hasa predicted PI of 4.77 (not
AMGGTGTGTCCCMTACCTGATAMTTCAMTACGGTMTGCAAAGACCGGAGATTTG 960
shown). A shorter and notcompletely sequenced (not shown) K G V S Q Y L N K F K Y G N A K T G D L 320
open reading frame extends 694 base pairs from the KpnI TGGGACGCCTTAGCAGATGCCTCTGGCMGGACGTTTGTTCGGTGA~GMCATCTGGACA 1020
W D A L A D A S G K D V C S V M N I W T 340
site.
A M C G T G T T G G T T T T C C A G T G T T A T C T G T C M G G M C A ~ G M C ~ T C A C A T T M C T 1080
Comparison of the AAPl DNA sequence and the predicted K R V G F P V L S V K E H X N K I T L T 360
protein sequence with sequences present in GenBank indi- CMCACCGTTATTTMGTACCGGTGATGATGTTAM~GAGGMGA~ACMCCATATACCCC 1140
cated that the putative AAPl gene product exhibits a high Q H R Y L S T G D V K E E E D T T I Y P 380

degree of sequence identity to several mammalian zinc ami- A T T T T G T T A G C T T T G M G A C A G T A C A G G T A T C G A ~ M C A C A T T G G T A T T ~ 1200

I L L A L K D S T G I D N T L V L N E K
C~
400
nopeptidases. The highest amino acid sequence identity was 1260
between the predicted AAPl protein and the mouse pre-B- 420
cell antigen B P I (39.5% identity over 505 amino acids) (6), 1320
440
rat zinc aminopeptidase (40.0% over 460 amino acids) (7),
1380
andhumanintestine zinc aminopeptidase (37.5% identity 460
over 483 amino acids) ( 5 ) . Lower but highly significant levels 1440
480
of amino acid sequence identity were found between Aapl
protein and human leukotriene A-4 hydrolase (25.5% identity 1500
500
over 357 aminoacids)(29)andE. coli aminopeptidase N 1560
(25.1% identity over 383 amino acids) (30). All of these 520

enzymes have been shown to exhibit aminopeptidaseactivity. 1581

526
During thesequence comparisons, five distinct regions were 1582 TGCACTTCTG G C M T G M MTGCAATCG ATTGCGGTAG AhATGTTTGA G G M T A C G C C
observed that are highly conserved in the mammalian pepti- 1642
1702
M T G G G M C AM C A G G C G A T
ATICTTCGGL:
ACCTGCACTG T T C M G G C A G TTGTTTTCM CACCGTTGCC
A C I I M C M T T A C C A M G A TTTTURCAT CTATCAhMC CCTGTTTCGT
dases and the predicted Aapl protein (Fig. 4). The regions 1162 C
G
i&- GLTFhTiFCT &
TG
<
A
iGC
TffiT
GT
CAGTT T G M G A C A M GIVICTTTTAG
1822 AAAGGACATT GACTTATCTG CTAGATCGTA C A G T T T T G M TCMGATTTT T A C A T C C C M
varybetween 15 and 35 aminoacidsinlength.Inthese
regions, the sequence identity between Aaplproteinand
mammalian aminopeptidases is very high, ranging from 53%
FIG. 3. DNA sequence of the AAPl gene and the predicted
(region I) to 93% (region 111) (Fig. 4). If conservative changes sequence of the Aapl protein. The bold boxes indicate regions of
are allowed, the similarity in these regions between the pre- high sequence identity with mammalian aminopeptidases (Fig. 4).
dicted Aapl amino acid sequence and the sequences of the The designated BglII site indicates thesite of HIS3 insertional
mammalian aminopeptidases approaches100%. No functions inactivation.
have been suggested for regions I, 11, and 111. Region IV has
a high sequence identity to the zinc-binding domainsof sev- leukotriene A-4 hydrolase, and the Aapl protein exhibit iden-
eral aminopeptidases (31) and region V shows homology to tities approaching 50% (not shown).
other peptidases with a putative proton donor a t tyrosine When the 4.4-kb EcoRI-KpnI AAPI probe is hybridized
residue 280 (32). with RNA isolated from cells grown to stationary phase in
WhentheAaplamino acidsequence is compared with rich, glucose-based medium (YPD), two transcripts of approx-
human leukotriene A-4 hydrolase or E. coli aminopeptidase imately 2.7 and 1.4 kbare observed(Fig. 5 ) . The larger
N, regions I, 11, and V are not conserved (not shown). In transcript was identified as U P 1 by reprobing the blot with
contrast, regions 111 and IV of E. coli aminopeptidase, human labeled fragments internal to the AAPi gene (not shown).
 Isolation
Characterization
and of the Yeaqt AAPl Gene 14313
during early exponentialgrowth (Fig. 5 ) .
T o determine whetherAAPl is an essentialgene, a disrup-
I II Ill IV v
COOH tion mutation was constructed (Fig. 2). Because clones ter-
- minating at theBgU site do not complement thespr5 muta-
50 AA tion, coding sequences flanking the site were assumed to be
essential for gene function. Wild type cells were transformed
with a 4.3-kbEcoRI-XbaI fragment containing AAPl dis-
rupted by the HIS3 gene (Fig. 2). The disruption was verified
by Southern blot analysis of chromosomal DNA from trans-
formed yeast cells (not shown). Haploid cells containing the
disruptionare viable, exhibitessentially wildt-ype growth
rates, and grow on nonfermentable carbon sources at 30 and
37 "C, but exhibit adecreased ability to accumulateglycogen,
which normally occurs at the diauxic shift, when glucose is
depleted from the medium (Fig. 6).
Because the BglII site, used for the HIS3 insertional inac-
tivation (Fig. 2) is downstreamof conserved regions I through
IV (Fig. 3), it was possible that a truncated butactive peptide
could be encoded by the disrupted gene. To evaluate this
I v nous*-1B p 1 QRVAEVIQHELAHQWFGNLVTMDWWEGLWLNEGFA
""s-vA"-v""" """ """" T DD possibility, we constructed a deletion/disruption mutation by
Human E--VT--A-------------IE"ND--ND--------
R a t zn E--VT--A-------------VD--N~-------- inserting LEU2 into HindIIIIBglII-digestedAAPl, creating a
deletion of the 5' end of AAPl (Fig. 2). Strains carrying the
AAPl FDAISYSKGSSLLRMIS
Uous. B P 1 --G------A-I---LO
LEU2 deletion/disruption, like strainscarryingthe HIS3
nman
~ """" ,?+-V"-L- disruption, are viable and able to grow on non-fermentable
Zn "S-T""A-V"-L-
carbon sourcesa t 30 and 37 "C and exhibitdecreased glycogen
FIG.4. Sequenceidentity between Aapl and mammalian accumulation (Fig. 6). We conclude from these results that
aminopeptidases. A, map of Aapl showing regionsof high homology AAPl is a non-essential gene whose gene product, whether
to mammalian aminopeptidases. Highly conserved regions are desig-
nated by Roman numerals. R, amino acid sequence and regions of directly or indirectly, acts as a positive regulator of glycogen
identity hetween Aapl andseveral mammalian aminopeptidases. The accumulation.
mammalian aminopeptidases shown are: Mouse BPI, mouse pre-B- AAPI was demonstrated to be a suppressor rather than an
cell antigen; Human, human intestine aminopeptidaseN; Rat Zn, rat allele of SPR-5 by sporulation of aapl spr5-27 diploids. Analy-
kidney zinc aminopeptidase. Residues shown are as follows: AAPI: sis of the segregants indicated that AAPl segregates inde-
region I, aa 21-50; region 11, aa 76-90; region 111, aa 155-169; region pendently of the s p ~ mutation
5 and, thus,is not allelic to spr5
IV, aa 186-220; region V, aa 275-291. Mouse BP1 (6): region I, aa
225-254; region 11, aa 284-298; region 111, aa 366-380; region IV, aa (not shown).Because a small increasein AAPI copy number,
397-431; region V, aa 489-505. Human aminopeptidase N ( 5 ) :region i.e. the AAPI gene carried on a centromeric vector, weakly
I, aa 208-237; region 11, aa 268-282; region 111, aa 349-363; region IV, suppressesthe spr,5 mutantphenotype, it is possible that
aa 380-414; region V, 472-488. Rat zinc aminopeptidase (7): region I, these two genes encode proteins that are functionally similar.
aa 204-233; region 11, aa 262-276; region 111, aa 344-358; region IV, T o further examine the role of AAPl on gene expression
aa 375-409; region V, aa 467-483. after the diauxic shift, control andaapl cells and controlcells
carrying pAAPl were transformed with the pWB206 plasmid
OD 600
(33), carrying the lacZ gene under the controlof the HSP70-
related SSA3 gene. SSA3 is a heat shock gene that is not
expressed in the culturecycle until after thediauxic shift and
whose transcriptioncontinuesintostationaryphase(10).
Overexpression of AAPl results in an approximately 8-fold
induction during the post-diauxic phase and 4-fold
a induction
AAPl+ - w C

3' 0RF-b D S 1 0 s p r l - 2 0r a s 2 -

FIG. 5. AAPl mRNA accumulation during growth of wild
type yeast to stationary phase in YPD. Total RNA was isolated
from cells at thedesignated culture densities. Twenty micrograms of
the total RNA was loaded per lane. A , transcripts detected with the
4.4-kh EcoRI-KpnI ( A A P I )probe. R, ethidium hromide-stained gel.
The smaller transcript alsohybridizes with the 1.8-kb XbaI-
KpnI probe containing the short open readingframethat
overlaps the KpnI site. AAPI mRNA is present throughout
theculture cycle butis more abundantinexponentially-
growing cells (Fig. 5 ) . The smaller transcript is presentonly
aapl::HIS3 aap1::LEUP pAAPl
FIG. 6. Glycogen accumulation in control, r-2, sprl-20,
and aapl cells. To visualize glycogen accumulation, cells were grown
on solid medium and exposed to iodine crystals as described under
"Materials and Methods." ras2 cells were used as a positive control
because they havebeenshown to h.yperaccumulate glycogen (40).
sprl-20 cells do not accumulate glycogen (D. R. Caprioglio, unpuh-
lished results) and were used as a negative control. The strains are
as follows: A A P l , DSIO; ra.92, dC302-26R; aap1::Hl.S:~. DC106; and
aapl::LEC12. DC126: sprl-20, DC20; pAAPI, DSIO carryingthe
pAAPl plasmid.
14314 Isolation and Characterization of the Yeast AAPl Gene
in stationaryphase,buthaslittle effect on SSA3p-lacZ rying a wild type AAPl gene are absent in strains carrying
expression after a heat shock (Fig. 7). Deletion of aapl has the aapl::HIS3 mutation or the uap1::LEUZ mutation and
relatively little effect on lac2 expressionfromthe SSA3 present in higher concentrations in cells carrying AAPl on a
promoterduringtheculture cycle butresultsin a slight multicopy plasmid (Fig. 8). Leucine aminopeptidase activity
dampening of the heat shockresponse. was present in both AAPl and aapl strains, indicating that
Due to the strong sequence similaritybetween the predicted the Aapl protein does not possessleucine aminopeptidase
Aapl protein sequence and well characterized aminopepti- activity (not shown). We conclude, based on the DNA se-
dases, we wanted to test whether the AAPl expression was quence identities and the loss of aminopeptidase activity, that
associated withaminopeptidaseactivity.Proteinextracts AAPl encodes an major alanine/arginine aminopeptidase in
from AAPI and aapl cells and cells carrying AAPl on a yeast.
multicopy vector were assayed for leucine, alanine, and argi- Finally, a novel band is apparent in extracts of proteins
nine aminopeptidase activity (Fig. 8). Protein extracts from from cells lacking a functional AAPl gene but is not detected
cells grown to late exponential phase were used for the initial inextractsfrom wild type cells visualized by Coomassie
characterization because AAPl mRNA is relatively abundant staining (Fig. 7). The novel band migratesmore slowly in
at this stage (Fig. 5). Two aminopeptidase activities separable nondenaturing gel electrophoresis than the putative Aapl
by nondenaturing gel electrophoresis were observed in the aminopeptidase. The presence of these bands in extracts from
parental strain (Fig. 8). The more rapidly migrating arginine cells lacking Aapl and theirabsence in control cells suggests
and alanine aminopeptidase activities present in strains car- the accumulationof specific proteins within the cell is affected
by Aapl protein activity.
DISCUSSION
b The AAPl gene was isolated basedon its ability to suppress
Controlstrain the growth defects on nonfermentable carbon sources exhib-
ited by spr5 strains. SPR5 is involved in regulation of genes
expressed during entry into stationary phase in yeast.’ Al-
though AAPl and SPR5 are not allelic, AAPI can suppress
the temperature sensitivityof spr5 mutants, when present on
either low or high copy number vectors. In addition, cells
carrying AAPl on a multicopy vector hyperaccumulate gly-
cogen, exhibit a n increased rate of growth on nonfermentable
carbon sources, and exhibit dramatic increases in transcrip-
Log PDS Stat HS tion of a gene normallyexpressedonly as cells approach
stationary phase. In contrast, cells carrying anAAPl disrup-
FIG. 7. &Galactosidase expression from an SSA3-lmZ fu- tion are less able than wild type to accumulateglycogen and
sion in control (DS10) and aapl cells (DC106) and in control
cells carrying the pAAPl plasmid. Assays were carried out as exhibit a slightly reducedtranscription of the heatshock gene
described under “Materials and Methods.” Cells were harvested a t SSA3 after a temperature upshift.
Am = 0.6-0.8,10-11, and 18-30, respectively for log, post-diauxic Analysis of AAPI nucleotide and predicted protein se-
shift (PDS),and stationary phase (Stat). Heat-shocked cells had quence indicates a high degree of homology with eucaryotic
been grown to anA m of 0.6-0.8. Experiments were done in triplicate aminopeptidases. In addition, mutants carrying disruptions
with maximum errors of 10%. a, 8-fold higher than control (DS10) in the AAPl gene lack alanine and arginine aminopeptidase
cells; b, 4-fold higher than control cells.
activity, whereas strains carryingAAP1 a on multicopy vector
exhibit increased alanine and arginine aminopeptidase activ-
ity. Basedonthese observations, we conclude that AAPl
encodes a protein with aminopeptidase activity.
-0 Aminopeptidases, when assayed with classical substrates,
exhibit a broadrange of substrate specificity. Of the 14
separate aminopeptidase and dipeptidyl aminopeptidase ac-
tivities identified in solubleextracts of S. cereuisiae (4), essen-
tially all exhibited activity with several substrates. For ex-
ample, leucine aminopeptidases from S. cereuisiae also hydro-
lyze lysine substrates (27, 31, 34). Aaplaminopeptidase
1 2 3 4 I L 3 4 ‘1 2- 3 4 activity hydrolyzes both alanine and arginine aminopeptidase
Coomassie Arginine Alanine substrates but isinactive against leucine aminopeptidase sub-
Stain strates.
Thefunction of aminopeptidasesin microorganisms is
FIG. 8. Aminopeptidaseactivity in control and aapl mutant
strains and strains carrying AAPl on a multicopy vector. poorly understood. It seems likely that at least some amino-
Soluble protein was extracted at late log phase as described under peptidases regulate intracellular proteolysis via the ubiquitin
“Materials and Methods.” + indicates samples from U P 1 cells. - pathway. NH2-terminal aminoacids have been shown to act
indicates samples from aapZ cells. Lane 1, DS10; lane 2, DC106 as signaling mechanismsto indicate short-lived orlong-lived
(aapl::HZS3); lane 3, DSlO carrying pAAP1; lane 4, DC126 proteins (35,36) and methionine aminopeptidases apparently
(aapZ::LEU2). Coomassie Stain, gel incubated with Coomassie Blue target proteins whose second amino acid acts as a stabilizing
stain after electrophoresis. Fifty micrograms of total protein loaded
per lane. Arrour indicates the location of a novel protein band in residue(35,36). However, for proteins with high turnover
extracts from aapl cells. Arginine, gel incubated with the substrate rates, some type of aminopeptidase activity must remove the
Arg-NH-naphthylamide. Three hundred micrograms of protein was terminal amino acid(s) to expose destabilizing residues. The
loaded per lane. Arrow indicates location of major aminopeptidase
activity. Alanine, same as section B but incubated with the Ala-NH- D. R. Caprioglio, C. Padilla, and M. Werner-Washburne, manu-
naphthylamide as a substrate. script in preparation.
 Isolation
Characterization
and ofYeast
the AAPl Gene 14315
specificity of the aminopeptidase encoded by AAPl is inter- 6. Wu, Q., Lahti, J. M., Air, G. M., Burrows, P. D., and Cooper, M. D. (1990)
Proc. Natl. A c d . Sci. U. S. A. 8 7 , 993-997
esting because arginine is oneof the most destabilizing NHZ- 7. Watt, V. M., and Yip, C. C. (1989) J . Biol. Chem. 264,5480-5487
terminal residues, whereasalanineis ahighly stabilizing 8. Chang, S.-Y. P., McGary, E. C., and Chang, S. (1989) J. Bacteriol. 1 7 1 ,
4071-4072
residue (3). Further work to determine the specificity of this 9. Chang, Y.-H., Teichert, U., and Smith, J. A. (1992) J. Biol. Chem. 2 6 7 ,
8007-801 1
arginine/alanine aminopeptidasewill be necessary to begin to 10. Werner-Washburne, M., Becker, J., Kosic-Smithers, J., and Craig, E. A.
define the role of this aminopeptidase in uiuo. (1989) J. Bacteriol. 171,2680-2688
11. Werner-Washburne, M., Stone, D. E., and Craig, E. A. (1987) Mol. Cell.
The positive effect of Aapl activity on glycogen accumula- Biol. 7,2568-2577
tion and expression from the SSA3p-lacZ fusion vector sug- 12. Cannon, J. F., and Tatchell, K. (1987) Mol. Cell. Biol. 7,2653-2663
13. Rose, M. D., Winston, F., and Hieter, P. (1990) Methods in Yeast Genetics:
gests that Aapl may be involved either directly or indirectly A Laboratory Course Manual, pp. 177-184, Cold Spring Harbor Labora-
in nutrient-sensing, since glycogen accumulates immediately tory Press, Cold Spring Harbor, NY
14. Algeri, A. A,, Bianchi, L., Viola, A. M., Puglisi, P. P., and Marmiroli, N.
prior to andduring glucose exhaustion andSSA3 is expressed (1981) Genetics 9 7 , 27-44
only after glucose exhaustion (10,37).Accumulation of AAPl 15. Naysmith, K. A,, and Tatchell, K.(1980) Cell 19,753-764
P. A,, Taylor, G. R., and Haynes, R. H.(1987) Nucleic Acids Res.
mRNA at OD,,, = 5 , aboutthetime glycogen begins t o 16. Lagosky,
1 5 , 10355-10371
accumulate (37), is consistent with such a role. We are cur- 17. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular C h i n A
Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Hartor,
rently examining changes in Aapl aminopeptidase activity NY
during growth to stationary phase and the interaction of this 18. Yanisch-Perron, C., Vieira, J., and Messing, J. (1985) Gene (Amst.) 3 3 ,
103-119
activity with other proteins known to affect glycogen accu- 19. Sikorski, R. S., and Hieter, P. (1989) Genetics 1 2 2 , 19-27
mulation, such asCAMP-dependent protein kinase, Bcyl (the 20. Jones, J. J., and Prakash, L. (1990) Yeast 6 , 363-366
21. Schmitt, M. E., Brown, T. A., and Trumpower, B. L. (1990) Nucleic Acids
regulatory subunit of CAMP-dependent protein kinase), Cyrl Res. 18,3091
(adenylate cyclase), Ras2, and Snfl kinase (required for glu- 22. Fourney, R.M.,Miyakoshi, J., Day,R. S., and Paterson, M. C. (1988)
Focus 1 0 . 5 4
cose derepression) (38, 39). Because this is the first amino- 23. Feinberg, A. P., and Vogelstein, B. (1984) Anal. Biochem. 1 3 7 , 266-267
peptidase implicated in the regulationof glycogen accumula- 24. Feinberg, A. P. and Vo elstein, B. (1983) A d . Biochem. 1 3 2 , 6-13
25. Devereux, J., Haeherli, 8.,and Smithies, 0. (1984) Nucleic Acids Res. 1 2 ,
tion, Aaplmay prove to be extremely valuable in understand- 387-395
ing
the
relationship between signaltransduction
and 26. Reynolds, A., and Lundblad, V. (1992) in Current Protocols in Molecular
Biology (Ausubel, F. M., Brent,R.,Kingston,R. E., Moore, D. D.
proteolysis in yeast. Seidman. J. G.. Smith. J. A.. and Struhl. K.. eds) Vol. 2. ~. _
DD. ' 13.6.1:
~ - ~ ~

13.6.4, John Wiley & Sons, Inc., New York ' ~'
27. Hirsch, H. H., Suarez-Rendueles, P., Achstetter, T., and Wolf, D. H. (1988)
Acknowledgments-We are extremely grateful to Mary Anne Nel- Eur. J. Biochem. 1 7 3 , 589-598
son, Gerry Johnston, and Helen Caprioglio and the members of our 28. Bollag, D. M., and Edelstem, S. J. (1991) in Protein Methods, pp. 143-160,
Wiley-Liss, Inc., New York
laboratoryfor helpful suggestions during the preparation of this 29. Funk,C.D.,Ridmark, O., andFu, J. Y. (1987) Proc. Natl. Acad.Sci.
manuscript. We also thank Will Boorstein for the pWB206 plasmid, U. S. A. 84, 6677-6681
Linda Riles for the generous gift of the ordered X library and help in 30. Bally, M., Murgier, M., and Lazdunski, A. (1984) Mol. Gen. Genet. 1 9 5 ,
~n7-61n
"",
mapping AAPl, and Harriet Yazzie for excellent technical assistance. "&"

31. Garcia-Alvarez, N., Cueva, R., andSuarez-Rendueles,P. (1991) Eur. J .

REFERENCES
1. Hirsch, H.H., Suarez-Rendueles, P., and Wolf, D. H. (1989) in Molecular
and Cell Biology of Yeasts (Walton, E. F., and Yarranton, G. T., eds) pp.
134-200, Van Nostrand Reinhold Co., Inc., New York
2. Jones, E. W. (1984) Annu. Reu. Genet. 18,233-270
3. Finley, D., and Chau, V. (1991) Annu. Rev. Cell Biol. 7,25-69
4. Achstetter, T., Ehmann, C., and Wolf,D. H. (1983)Arch. Biochem. Biophys.
2 2 6 , 292-305
5. Olsen, J., Cowell, G. M., K~nigsh@fer, E., Danielsen,E.M.,M@ller,J.,
Laustsen, L., Hansen, 0. C., Welinder, K. G., Enberg, J., Hunziker, W.,
Spiess, M., Sjostrom, H., and Noren, 0. (1988) FEBS Lett. 2 3 8 , 307-
314
Biochem. 202,993-1002
32. Kester, W. R., and Matthews, B. W. (1977) J . Biol. Chem. 252 7704-7710
33. Boorstein, W. R., and Craig, E. A. (199Ob) Mol. Cell. Biol. 1 0 , g262-3267
34. Cueva, R., Garcia-Alvarez, N., and Suarez-Rendueles,P. (1989) FEBS Lett.
2 5 9 , 125-129
35. Gonda, D. K., Bachmair, A., Wunning, I., Tobias, J. W., Lane, W. S., and
Varshavsky, A. (1989) J . Biol. Chem. 2 6 4 , 16700-16712
36. Bachmalr, A,, Finley, D., and Varshavsky, A. (1986) Science 234,179-186
37. Lillie, S. H., and Pringle, J. R. (1980) J . Bacteriol. 1 4 3 , 1384-1394
38. Broach, J. H., and Deschenes, R. J. (1990) Adu. Cancer Res. 54.79-139
39. Thompson-Jaeger, S., Fransois, J., Gaughran,J. P., and Tatchell,K. (1991)
Genetics 129,697-706
40. Tatchell, K., Robinson, L. C., and Breitenbach, M. (1985) Proc. Natl. Acad.
Scr. U. S. A. 82,3785-3789

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