Sit4p Protein Phosphatase Is Required for Sensitivity of
Saccharomyces cerevisiae
to
Kluyveromyces lactis
Zymocin
Daniel Jablonowski,* Andrew R. Butler,
†,1Lars Fichtner,* Donald Gardiner,
†Raffael Schaffrath*
and Michael J. R. Stark
†*Institut fu¨r Genetik, Martin-Luther Universita¨t Halle-Wittenberg, D-06120 Halle (Salle), Germany and†Division of Gene Regulation & Expression, School of Life Sciences, University of Dundee, Dundee DD1 5EH, United Kingdom
Manuscript received June 25, 2001 Accepted for publication September 18, 2001
ABSTRACT
We have identified twoSaccharomyces cerevisiaegenes that, in high copy, confer resistance toKluyveromyces lactiszymocin, an inhibitor that blocks cells in the G1phase of the cell cycle prior to budding and DNA replication. One gene (GRX3) encodes a glutaredoxin and is likely to act at the level of zymocin entry into sensitive cells, while the other encodes Sap155p, one of a family of four related proteins that function positively and interdependently with the Sit4p protein phosphatase. IncreasedSAP155dosage protects cells by influencing the sensitivity of the intracellular target and is unique among the fourSAPgenes in conferring zymocin resistance in high copy, but is antagonized by high-copySAP185or SAP190. Since cells lackingSIT4or deleted for bothSAP185andSAP190are also zymocin resistant, our data support a model whereby high-copy SAP155promotes resistance by competition with the endogenous levels of SAP185and SAP190expression. Zymocin sensitivity therefore requires a Sap185p/Sap190p-dependent function of Sit4p protein phosphatase. Mutations affecting the RNA polymerase II Elongator complex also conferK. lactis zymocin resistance. Sincesit4⌬ andSAP-deficient strains share in common several other phenotypes associated with Elongator mutants, Elongator function may be a Sit4p-dependent process.
K
ILLER strains of the yeastKluyveromyces lactissecrete other chitinases and chitin-binding proteins (Butleret al.1991a). Recent work has demonstrated that muta-a protein toxin or zymocin thmuta-at inhibits theprolif-eration of several different yeasts including Saccharo- tions affecting at least five genes encoding components of the RNA polymerase II Elongator complex lead to myces cerevisiae (Stark et al. 1990; Schaffrath and
Breunig2000). In the presence of K. lactis zymocin, zymocin resistance (Frohloffet al.2001). Elongator is a multisubunit complex that binds to the elongating sensitive S. cerevisiae cells become blocked in the G1
phase of the cell division cycle prior to budding and form of RNA polymerase II (RNAPII:Oteroet al.1999;
Wittschiebenet al.1999;Fellowset al.2000) and loss with unreplicated DNA (White et al. 1989; Butleret
al. 1991c), suggesting that the zymocin acts to inhibit of Elongator function causes a range of phenotypes including delayed activation of genes under changing one or more of the events normally triggered at Start
growth conditions, hypersensitivity to 6-azauracil and and that are required for G1exit. Although the zymocin
caffeine, slow growth, and temperature sensitivity (
Ot-is a heterotrimeric protein, intracellular expression of
eroet al.1999;Frohloffet al.2001). However, Elonga-just the␥-subunit is sufficient to promote the G1arrest
tor itself is dispensable; so although zymocin inhibition phenotype (Tokunagaet al.1989;Butleret al.1991b).
is an Elongator-dependent process, the zymocin cannot The two larger subunits are probably involved in entry
simply be acting to block Elongator function. of the zymocin into the cell, a process that involves an
The execution of Start requires the Sit4p protein phos-interaction between the␣-subunit and cell wall chitin.
phatase and temperature-sensitivesit4mutants to arrest Thus chitin-deficient mutants are zymocin resistant
in G1 prior to bud emergence and DNA replication (TakitaandCastilho-Valavicius1993;Jablonowski
(Sutton et al. 1991; Fernandez-Sarabiaet al. 1992). et al.2001) and the␣-subunit has a domain that shows
This requirement is at least in part because Sit4p is in vitrochitinase activity and has sequence similarity to
needed for proper expression of G1 cyclins such as CLN1,CLN2, andPCL1. However, while ectopic expres-sion ofCLN2 from a Sit4p-independent promoter can
Corresponding author:Michael J. R. Stark, Division of Gene
Regula-relieve the block to DNA replication in sit4-102 Ts⫺
tion & Expression, School of Life Sciences, University of Dundee,
Dundee DD1 5EH, United Kingdom. cells, these cells still remain largely unbudded and thus E-mail: m.j.r.stark@dundee.ac.uk
blocked for bud emergence (Fernandez-Sarabiaet al. 1Present address:Department of Biochemistry, School of Biological
1992). Thus, independently of its effect onCLN2
expres-Sciences, University of Leicester, University Road, Leicester LE1 7RH,
United Kingdom. sion, Sit4p is also needed for other events required for
ated from CY4029 in the same manner. LFY5 and LFY6 were G1 exit. The phenotype ofsit4 mutants is complicated
constructed from AY925 by one-step disruption using the PCR by its dependence on a second polymorphic gene,SSD1
primers described previously (Frohloffet al.2001). (Suttonet al.1991). Inssd1-dstrains deletion ofSIT4 General methods:All yeast growth media and general yeast is lethal, but inSSD1-vstrainssit4deletion is tolerated, methods were as described byKaiseret al.(1994). Yeast trans-formation was carried out according to Gietzet al.(1992). although it leads to a G1 delay and a slow growth rate
When direct selection for theleu2dmarker gene was employed, that is not improved by ectopicCLN2expression (
Fer-the selective plates were supplemented with 0.0075% (w/v)
nandez-Sarabiaet al.1992). Sit4p associates in a
cell-Bacto-yeast extract. To test multiple strains for growth pheno-cycle-dependent manner with the members of a protein types on agar plates, strains were first grown on plates with family termed the SAPs (Suttonet al.1991) and dele- selection for all markers and then colonies of similar size were tion of all fourSAPgenes (SAP4,SAP155,SAP185, and resuspended in fresh medium at the same cell density (ⵑ106 cells/ml). Culture samples (ⵑ5l) together with three 10-fold SAP190) confers the same phenotype as loss of SIT4
serial dilutions were spotted onto plates using a multipronged (Lukeet al.1996). A variety of evidence shows that the
inoculating manifold (Dan-Kar). General recombinant DNA SAPs and Sit4p function in an interdependent manner, procedures were carried out as described by
Sambrooket al.
leading to models whereby the SAPs are either positive (1989). DNA sequencing was performed manually using the activators of Sit4p phosphatase or Sit4p effectors (Luke Sequenase version 2.0 kit (United States Biochemical, Cleve-land) and double-stranded plasmid DNA templates. A combi-et al. 1996). Sit4p also binds Tap42p, an element in
nation of specific subclones and synthetic oligonucleotide the TOR signaling pathway; on nutrient starvation or
primers allowed determination of the sequence ofSAP155on inhibition of the TOR kinases by rapamycin, Tap42p
both strands. To identify pMA3a clones encoding tRNAglu3 se-dissociates from Sit4p (Di ComoandArndt1996). Nu- quences, plasmid DNA was digested withEcoRI andSalI and trient starvation or treatment with rapamycin leads to Southern blot analysis was performed using the 435-bp BsaAI-HindIII fragment from pYF1 (Butler et al. 1994). Similar a variety of responses including a G1 arrest, reduced
analysis was performed using a 550-bp fragment of KTI12 translational initiation, reduced ribosome biosynthesis,
excised from pJHW27 to identifyKTI12-encoding clones. In and changes to the amino acid transporters expressed
each case, radiolabeled probes were generated using random on the cell surface (seeSchmidtet al.1998;Cardenas hexamer priming (Feinberg and Vogelstein 1983). RNA
et al.1999). Dissociation of Sit4p from Tap42p has been isolation and RT-PCR were both carried out essentially as proposed to trigger dephosphorylation of at least some described byFrohloffet al. (2001) using the following primer pairs:TOT1(5⬘-CTTGGTGTATGAAACTCGCG-3⬘ and 5⬘-TTC of the proteins that act as effectors of the TOR pathway
TTACCTCTGCCAGTACC-3⬘),TOT2(5⬘-AACCTGATGAGACT and that mediate some of these responses (Schmidtet
TCAGGC-3⬘ and 5⬘-CAAACCTAACACAGGAACGG-3⬘), TOT3 al.1998;BeckandHall1999). (5⬘-TCAGTCCTTGTACGAAGACG-3⬘ and 5⬘-ATAAGCTCGAC We previously isolated two genes that conferK. lactis CTGATCTGG-3⬘),TOT4(5⬘-TCCGGTATCAACTTCACTGC-3⬘ zymocin resistance when present specifically on high- and 5⬘-CTTGTTCCGTTACTTACCCC-3⬘), andHHT1(5⬘-AGC AAGAAAGTCCACTGGTG-3⬘ and 5⬘-GAATGGCAGCCAAGT copy plasmids (Butler et al. 1994). One of these
TGGTA-3⬘). (KTI12) had previously been defined by recessive
muta-Plasmids:Table 2 summarizes plasmids used in this study. tions that also lead to zymocin resistance. Since either
TheTAP42fragment in pDJ14 was obtained by PCR amplifica-loss of KTI12 function or high-copy KTI12 conferred tion followed by in vivo gap repair (to recover the coding zymocin resistance, we hypothesized that Kti12p could region from the genome) and complemented the lethality of atap42deletion strain (not shown). The high-copy library used be part of a protein complex required for zymocin
ac-in this study comprised partial Sau3A fragments from yeast tion that was perturbed by excess Kti12p (Butleret al.
genomic DNA (6–10 kb) inserted into the vector pMA3a (leu2d 1994). Consistent with this idea,KTI12has recently been
2) and has also been described previously (Crouzet and shown to be allelic with TOT4, a component of the Tuite1987). pARB19 was constructed in two stages: the large Elongator complex (Frohloff et al. 2001). Here we EcoRI fragment from the pARB106 insert was first ligated into describe the isolation ofGRX3andSAP155 (encoding theEcoRI site of YEplac181 and then theSalI fragment from this construct was replaced with theSalI fragment of pARB15 one of the Sit4p-associated SAPs) as additional genes
(Table 2) such that the bulk of the original insert of pARB106 that promote zymocin resistance when present in high
was reconstructed. pDG8 was made by first cloning the 1.96-copy. In addition, we show that cells lackingSIT4 are kbXbaI fragment of pARB100 (encoding the promoter and both refractory toK. lactis zymocin and share many of 5⬘half ofSAP155) into theXbaI site of YEplac181 such that the other phenotypes of Elongator mutants, placing theSAP155open reading frame (ORF) read in the opposite direction to the lacZ␣-fragment. TheSalI-PstI interval from Sit4p within a pathway required for zymocin action and
the insert was then removed and replaced with the SAP155 linking Sit4p to Elongator function.
SalI-PstI fragment from pDG6, reinstating a completeSAP155 gene. To make asap155::HIS3deletion allele, pDG8 was di-gested withBclI to excise the bulk of theSAP155coding region, MATERIALS AND METHODS
which was replaced by the 1.3-kb HIS3 BamHI fragment of YDpH (Berbenet al.1991). This resulted in the deletion of
Strains:All yeast strains used in this study are listed in Table
codons 85–999 in SAP155and yielded pDG11 and pDG12, 1. LL20-2 was generated from LL20-1 by transformation with
which differ in the orientation of theHIS3insert. pHO1.5 (a YEp vector carrying theHOandURA3genes; M.
K. lactiszymocin methods:Killer eclipse assays for zymocin Pocklington) to promote mating-type switching, followed by
sensitivity were performed as described previously (Kishida
5-fluoroorotic acid-induced plasmid loss and verification of
et al.1996), usingK. lactisstrains AWJ137 (zymocin producer) mating type by crossing with tester strains (Herskowitzand
dilu-TABLE 1
Yeast strains
Strain Genotypea Source
K. lactis
AWJ137 MATaleu2 trp1[pGKl1⫹pGKl2⫹] Ka¨mperet al. (1991) NK40 MAT␣ade1 ade2 leu2[pGKl1⬚pGKl2⫹] Gungeet al.(1981) S. cerevisiae
AY925 MATaW303ssd1-d2 Kim Arndt
CY3938 MATaW303sit4⌬::HIS3 SSD1-v1 Lukeet al.(1996) CY4029 MATaW303SIT4 SSD1-v1 Lukeet al.(1996)
CY4029␣ MAT␣W303SIT4 SSD1-v1 This study
CY5220 MATaW303sap4::LEU2 sap155::HIS3 SSD1-v1 Lukeet al.(1996) CY5224 MATaW303sap185::ADE2 sap190::TRP1 SSD1-v1 Lukeet al.(1996) CY5236 MATaW303sap4::LEU2 sap155::HIS3 Lukeet al.(1996)
sap185::ADE2 sap190::TRP1 SSD1-v1
DJY8 MAT␣W303sap4::LEU2 SSD1-v1 CY4029␣ ⫻CY5236
DJY9 MAT␣W303sap155::HIS3 SSD1-v1 CY4029␣ ⫻CY5236
CY4917 MATaW303sap185::ADE2 SSD1-v1 Kim Arndt CY4380 MATaW303sap190::TRP1 SSD1-v1 Kim Arndt
DJY10 MAT␣W303sap4::LEU2 sap185::ADE2 SSD1-v1 CY4029␣ ⫻CY5236 DJY11 MAT␣W303sap4::LEU2 sap190::TRP1 SSD1-v1 CY4029␣ ⫻CY5236 DJY12 MAT␣W303sap155::HIS3 sap185::ADE2 SSD1-v1 CY4029␣ ⫻CY5236 DJY13 MAT␣W303sap155::HIS3 sap190::TRP SSD1-v1 CY4029␣ ⫻CY5236 DJY14 MAT␣W303sap155::HIS3 sap185::ADE2 sap190::TRP1 SSD1-v1 CY4029␣ ⫻CY5236 DJY15 MAT␣W303sap4::LEU2 sap185::ADE2 sap190::TRP1 SSD1-v1 CY4029␣ ⫻CY5236 DJY16 MAT␣W303sap4::LEU2 sap155::HIS3 sap190::TRP1 SSD1-v1 CY4029␣ ⫻CY5236 DJY17 MAT␣W303sap4::LEU2 sap155::HIS3 sap185::ADE2 SSD1-v1 CY4029␣ ⫻CY5236 LA MAT␣leu2-3 leu2-112 his3-11 his3-15 ura3::pARB1Gal⫹ From LL20-1
LFY5 MATaW303tot3::TRP1 SSD1-v1 This study
LFY6 MATaW303kti12::TRP1 SSD1-v1 This study
LL20 MAT␣leu2-3 leu2-112 his3-11 his3-15Gal⫹ NCYC1445b
LL20-1 MAT␣leu2-3 leu2-112 his3-11 his3-15 ura3Gal⫹ Butleret al.(1994) LL20-2 MATaleu2-3 leu2-112 his3-11 his3-15 ura3Gal⫹ Vera Martin
LL20-3 MATa/MAT␣LL20 LL20-1⫻LL20-2
LY MAT␣leu2-3 leu2-112 his3-11 his3-15 ura3::YIplac211 Gal⫹ From LL20-1 TS54-1A MATaleu2-3, 112 ura3-52 rme1 HMLatap42::kanMX M. Hall
Gal⫹[YCplac111::tap42-11]
aW303 genetic background isade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1 can1-100Gal⫹and, normally,ssd1-d2. bNational Collection of Yeast Cultures, Colney, Norwich, UK.
tions of strains (grown under selection for plasmids as appro- gene previously defined by a recessive, zymocin-resistant priate) were spotted out onto YPD agar with or without culture mutation and recently shown to encode a component of supernatant from AWJ137 as a source of toxin [65% (v/v)
the RNAPII Elongator complex (Frohloffet al.2001). unless otherwise stated]. Growth was compared after 2 days
Since the previous screen was far from saturated we incubation at 26⬚. To test plasmids for conferring resistance
to intracellular expression of the zymocin␥-subunit, they were examined a different high-copy library for additional introduced into strains LA and LY (Table 1) with selection sequences that could confer zymocin resistance and that on SD medium with appropriate supplements, and then
trans-might, likeKTI12, also correspond to genes defined by formants were tested for growth on S agar containing 2%
our collection of zymocin-resistant mutations (Butler
raffinose and 2% galactose. Other strains were similarly tested
et al.1994). To avoid obtaining spontaneous zymocin-by introduction of pLF16 (encoding aGALpromoter-zymocin
␥-subunit fusion) or YCplac111 (as a control). resistant mutants among our transformants, we con-ducted the present screen in a homozygous diploid strain (LL20-3) transformed with a yeast genomic library RESULTS in pMA3a, selecting Leu⫹transformants directly. Since
pMA3a carries the leu2d marker it is expected to be Isolation of sequences conferring resistance to K.
present at very high copy number when transformants
lactiszymocin in high copy:We previously isolated two
are grown under selection for Leu⫹(Erhartand
Hol-S. cerevisiaegenes that conferred resistance to K. lactis
lenberg1983). Cells fromⵑ12,500 independent trans-zymocin when introduced in high copy into sensitive
formants (ⵑ1.5 genome equivalents) were recovered strains ofS. cerevisiae(Butleret al.1994). One of these
TABLE 2
Plasmids
Name Description Source
pARB1 1.3-kbEcoRI-SalI fragment carrying theGAL1-zymocin␥-subunit gene Butleret al.(1994) fusion in YIplac211
pARB7 4.8-kbEcoRI-SalI fragment from pARB106 in YEplac181 This study pARB15 3.5-kbSalI fragment from pARB106 in YEplac181 This study pARB18 8.2-kbEcoRI-SalI fragment carryingSAP155in YCplac111 This study pARB19 8.2-kbEcoRI-SalI fragment carryingSAP155in YEplac181 This study pARB22 5.9-kbEcoRI-SalI fragment of pARB100 in YCplac111 This study pARB23 6.5-kbEcoRI-SalI fragment of pARB100 in YEplac181 This study pARB31 3.0-kbPvuII fragment of pAB106 in YEplac181 This study pARB36 3.8-kbPvuII fragment of pAB106 in YEplac181 This study pARB100 5.25-kb fragment carryingGRX3/tRNAglnin pMA3a This study pARB106 8.4-kb fragment carryingSAP155in pMA3a This study
CB337 SIS2in YEp24 Kim Arndt
CB1317 ADH1-CLN2inLEU2-2 Kim Arndt
CB1418 ADH1-PCL1inLEU2-CEN Kim Arndt
CB2606 SAP190in YEp24 Kim Arndt
CB2643 SAP155in YEp24 Kim Arndt
CB2645 sap155fsin YEp24 (frameshift mutation inSAP155) Kim Arndt
CB2819 SAP185in YEp24 Kim Arndt
CB2925 SAP4in YEp24 Kim Arndt
pDG6 3.3-kbPsp1406I fragment ofSAP155in theClaI site of pBluescript II SK⫹ This study pDG8 3.8-kbXbaI-Psp1406I fragment ofSAP155in YEplac181 This study
pDG11, 12 sap155::HIS3versions of pDG8 This study
pDG14 0.62-kbXhoI-BamHI fragment of pARB100 in YEplac181 (SalI-BamHI) This study carrying tRNAgln
pDG15 1.4-kbSpeI-XhoI fragment of pARB100 in YEplac181 (XbaI-SalI) carrying This study GRX3and tRNAgln
pDJ14 1.8-kbEcoRI-PstI fragment carryingTAP42in YEplac195 This study
pJHW27 2.9-kbDraI fragment carryingKTI12in YEplac181 Butleret al.(1994) pLF16 1.45-kbEcoRI-HindIII fragment of pARB1 (carrying theGAL1-zymocin This study
␥-subunit gene fusion) in YCplac111
YCplac33 CEN4 ARS1 URA3yeast shuttle vector GietzandSugino(1988) YCplac111 CEN4 ARS1 LEU2yeast shuttle vector GietzandSugino(1988) YEp24 2URA3yeast shuttle vector Botsteinet al.(1979) YEplac181 2LEU2yeast shuttle vector GietzandSugino(1988) YEplac195 2URA3yeast shuttle vector GietzandSugino(1988) pYF1 1.9-kbHindIII-BamHI fragment carrying tRNAglu3 in YEplac181 Butleret al.(1994)
a range of concentrations of crudeK. lactiszymocin in and tE(UUC)E2:Butleret al.(1994)]. Representative clones from each of the two remaining categories of YPD agar. From 5.5 ⫻ 106 cells thus screened, ⵑ200
clones that could grow at concentrations of 0.25% crude clone (pARB100 and pARB106) were therefore charac-terized further.
zymocin or above were selected. From these, 50 plasmids
were recovered that conferred resistance to 1.8% crude High-copyGRX3confersK. lactiszymocin resistance: The entire insert of pARB100 was transferred from zymocin when reintroduced into LL20-3. Restriction
mapping allowed these plasmids to be classified into pMA3a into both YCplac111 and YEplac181 as an EcoRI-SalI fragment (see Figure 1A). Since the YEplac181 de-several different categories.
Since we had already shown that tRNAglu3 genes and rivative (pARB23) conferred zymocin resistance when transformed into LL20, we concluded that the extra KTI12 could each confer zymocin resistance in high
copy, clones from the current screen were examined by high copy number of the original isolate (pARB100) imposed byleu2dselection was not essential for the insert Southern blot analysis using tRNAglu
3 andKTI12probes
(not shown). This allowed identification of fourKTI12 to function as a multicopy resistance determinant. The low-copy construct (pARB22) was introduced into a rep-clones and three rep-clones encoding two tRNAglu3 loci
[tE(UUC)GL2 and tE(UUC)ER3: seeHaniandFeld- resentative strain from each of the 13 complementation groups ofK. lactiszymocin-resistant mutants described
mann(1998)], which were distinct from the two other
classes of zymocin-resistant mutant, namely those in which the zymocin’s intracellular target is altered to render it insensitive and others that are resistant to exogenous zymocin but still contain a sensitive intracel-lular target (Butler et al. 1994). Since presence of pARB100 did not protect cells from intracellular expres-sion of the zymocin␥-subunit (not shown), we conclude that high-copyGRX3is unlikely to function at the level of the zymocin’s intracellular target but is more likely to operate by impairing entry of native zymocin. By comparison, high-copySAP155(see below), tRNAglu3 , or KTI12(Butleret al.1994) can each confer protection from zymocin␥-subunit expression.
The zymocin resistance determinant on pARB106 is
SAP155:DNA sequencing demonstrated that pARB106
encoded two complete genes on chromosome VI, YFR039c andSAP155.SAP155is one of four relatedSAP
Figure 1.—Characterization of clones that promote
zy-genes (SAP4,SAP155,SAP185, andSAP190) that show mocin resistance in high copy. Maps indicate key restriction
a functional relationship with the Sit4p protein phos-sites in both the insert (—) and the flanking pMA3a vector
sequences ( ), together with the genes encoded by the insert phatase (Luke et al. 1996). pARB106 subclones were ( ). Below the maps, open boxes ( ) indicate subclones made in YEplac181 (Figure 1B) and tested for their that failed to confer zymocin resistance in high copy, while
ability to confer zymocin resistance. Neither the EcoRI-solid boxes ( ) indicate subclones that continued to confer
SalI nor theSalI-SalI fragments derived from the insert zymocin resistance when inserted into YEplac181. (A)
could confer zymocin resistance, although a reconstruc-pARB100 subclones. The inset compares the zymocin
resis-tance of CY4029 (W303SSD1-v1) carrying pARB100, pDG14, tion of these two fragments (pARB19) was completely and pDG15 by plating 10-fold serial dilutions of cells onto functional in the assay. This demonstrated that the extra YPD agar with or withoutK. lactiszymocin with a high-copy
high copy number of pMA3a was not essential for con-tRNAglu3 clone (pYF1) as a control. (B) pARB106 subclones.
ferring resistance and also ruled out YFR039c as the resistance determinant. In contrast,SAP155 alone was capable of conferring resistance when subcloned into tested for zymocin resistance in each case. Since
comple-mentation was not observed in any instance (not shown), YEplac181 (Figure 1B, pDG8), confirming it as the resis-tance determinant on pARB106. Consistent with this we concluded that pARB100 does not correspond to
any of the previously describedKTI loci. pARB22 also conclusion, pDG8 derivatives in which theSAP155 cod-ing region was replaced by HIS3 or a construct that failed to confer resistance on wild-type strains,
demon-strating that the resistance determinant was required carried a frameshift mutation in SAP155 were unable to confer zymocin resistance (not shown). Unlike specifically in high copy.
DNA sequencing revealed that pARB100 carried an pARB100, pARB106 could protect cells from growth arrest by intracellular expression of the zymocin␥ -sub-insert from chromosome IV that carried three genes
(Figure 1A). Subclones from pARB100 were made in unit, allowing growth of strain LA on galactose-con-taining medium (not shown). Thus, unlikeGRX3, high-YEplac181 and tested for their ability to conferK. lactis
zymocin resistance (Figure 1A). A YEplac181 subclone copySAP155 confers zymocin resistance at the level of its intracellular target. The pARB19 insert was also carryingGRX3(pDG15) was an effective zymocin
resis-tance determinant (Figure 1A, inset). Since this region cloned into YCplac111 and introduced into the repre-sentative K. lactis zymocin-resistant mutant strains de-encodes a tRNAgln in addition to GRX3 and because
tRNAglu
3 genes confer zymocin resistance in high copy, scribed above. This construct (pARB18) failed either to confer zymocin resistance on wild-type strains or to we tested a YEplac181 subclone carrying the tRNAgln
alone (pDG14). pDG14 was unable to confer zymocin complement zymocin resistance in any of the mutants (not shown), indicating thatSAP155is needed in high resistance (Figure 1A, inset), thereby ruling out the
tRNAgln as the critical factor. copy and that it does not correspond to any of the previously describedKTIgenes.
GRX3is one of three related yeast glutaredoxin genes
that differ from classical glutaredoxins by having a single In the process of identifying SAP155as the zymocin resistance determinant, a large section of the pARB106 cysteine residue at the putative active site (Grant2001).
To determine the level at which GRX3 functions, insert surrounding theSalI site was sequenced on both strands (EMBL accession no. AJ318331). The sequenced pARB100 was tested for its ability to protect yeast cells
from growth arrest by conditional intracellular expres- region (corresponding to chromosome VI bases 233710– 237771) differs in six positions from the current se-sion of the zymocin␥-subunit from theGALpromoter.
Figure 2.—Alignment of the predicted terminal sequences of the four SAP proteins. The similarity of the amino-terminal extension to Sap155p predicted by this and other work (Lukeet al.1996) to Sap4p, Sap185p, and Sap190p is indicated. • indicates the start of the Sap155p ORF predicted from current database entries. The arrow and circled amino acid denote the position of the frameshift (in the glycine codon) that extends the ORF as shown.
Database. One of these differences (deletion of G234450 dent on the SSD1 locus, we also compared isogenic ssd1-dandSSD1-vstrains for zymocin sensitivity and for in our sequence) extends the SAP155 open reading
frame by 97 codons, in agreement withLukeet al.(1996). the effect of high-copySAP155.Bothssd1-dandSSD1-v strains are inhibited by zymocin (Figure 4), although The putative amino-terminal extension generated by
this change shows strong similarity to the amino-termi- ssd1-d cells are slightly more zymocin sensitive (Figure 4). Furthermore, high-copySAP155 could promote zy-nal sequences of the other three SAPs (Sap4p, Sap185p,
and Sap190p; Figure 2) and is therefore likely to be mocin resistance in both genetic backgrounds (Figure 4), whileTAP42had no effect in either (Figure 3B). correct.
OnlySAP155and none of the otherSAPgenes func- Sit4 phosphatase and the SAP185/SAP190 family are
required for zymocin sensitivity: We next tested the tion as a high-copy zymocin resistance determinant:
SAP4,SAP155,SAP185, andSAP190encode a family of zymocin sensitivity of isogenicSSD1-vstrains that were either wild type or deleted forSIT4or various combina-related proteins that function interdependently with the
protein phosphatase Sit4p, each of which can partially tions of SAP genes. As shown in Figure 5, deletion of no singleSAPgene conferred significant zymocin resis-suppress the Ts⫺growth defect ofsit4-102 ssd1-dstrains
(Lukeet al.1996). The four SAP proteins fall into two tance. In comparison, loss ofSIT4rendered cells resis-tant to zymocin. High-copySAP155 was unable to pro-groups based on sequence similarity, with Sap4p and
Sap155p in one group and Sap185p and Sap190p in mote additional zymocin resistance in sit4⌬ strains (Figure 3C), consistent with high-copy SAP155 acting the other. These two groups are functionally distinct
since, for example, Sap185p and Sap190p (but not through Sit4p rather than by some alternative route. Although loss of singleSAPgenes was without effect on Sap4p and Sap155p) are together required for a Sit4p
function that is essential in the absence ofBEM2(Luke zymocin sensitivity, the doublesap185⌬sap190⌬strain was as resistant to zymocin as thesit4⌬ strain, and all et al.1996). Although Sap4p could not be found in Sit4p
immune precipitates, Sap155p, Sap185, and Sap190p other multipleSAPdeletion strains that also lacked both SAP185andSAP190were zymocin resistant (Figure 5). each form complexes separately with Sit4p and, by
sev-eral criteria, cells lacking all fourSAPgenes behave very In comparison, cells lacking both SAP4 and SAP155 were fully zymocin sensitive. Like high-copy SAP155, likeSIT4-deficient cells (Lukeet al. 1996). We therefore
next tested YEp24 clones of all fourSAPgenes for ability knockout strains lacking SIT4 or both SAP185 and SAP190promoted zymocin resistance at the level of the to confer zymocin resistance. Unlike SAP155, each of
the other three SAPgenes failed to protect cells from zymocin’s intracellular target, since these mutant strains were also resistant to intracellular expression of the inhibition by zymocin (Figure 3A).
In addition to forming complexes with the SAPs, Sit4p zymocin␥-subunit from theGALpromoter (Figure 6). Thus zymocin sensitivity requires a Sap185p/Sap190p-also interacts with Tap42p in a TOR-dependent and
rapamycin-sensitive manner (Di Como and Arndt mediated Sit4p function.
Since the SAPs have been shown to compete with 1996). However, high-copyTAP42also failed to promote
zymocin resistance (Figure 3B). We also tested whether each other for binding to Sit4p (Lukeet al.1996), our data suggested a simple model for how high-copy other genes that improve the growth defect ofsit4
mu-tants in high copy or when overexpressed could pro- SAP155might promote zymocin resistance: higher levels of Sap155p in strains with extra copies ofSAP155might mote zymocin resistance. However, neither high-level
expression ofCLN2orPCL1(from theS. cerevisiae ADH out-compete Sap185p and Sap190p for binding to Sit4p, thereby opposing the Sap185/190p-dependent Sit4p promoter) nor high-copySIS2conferred zymocin
resis-tance (not shown). Thus the effect of SAP155 in this function required for zymocin sensitivity. Such a model predicts that extra copies ofSAP185orSAP190should assay is highly specific.
Figure3.—Effect of high-copy genes on zy-mocin sensitivity. In A, C, and D, 10-fold serial dilutions (right to left) of either CY4029 (W303 SSD1-v1) or CY3938 (sit4::HIS3) transformed with control URA3 vectors or 2-URA3 plas-mids carrying SAPgenes were replica plated onto YPD (left) or YPD containing zymocin (middle) or assessed using the eclipse assay (A and D, right). In the latter assay, the size of the growth inhibition halo around theK. lactis zymocin-producing colony, inoculated on the edge of a spot of S. cerevisiae cells, indicates the level of sensitivity/resistance shown by the latter. In B, the eclipse assay was used to com-pare sensitivity of ssd1-d2(AY925) and SSD1-v1(CY4029) strains carrying high-copyTAP42 with untransformed strains.
confirms this prediction, showing that extra copies of tot3⌬ control strain. Surprisingly, the sap4⌬ sap155⌬ double mutant was more hypersensitive to 6-azauracil eitherSAP185 or SAP190 greatly reduce the ability of
high-copySAP155to promote zymocin resistance. despite failing to confer temperature sensitivity or zy-mocin resistance. Because SIT4 was first identified Loss of Sit4p and SAP function share common
pheno-types with Elongator mutations:Since mutations in com- through mutations that affect RNAPII transcription of a variety of genes (Arndt et al. 1989), it is possible ponents of the RNAPII Elongator complex cause
zy-mocin resistance, we next examined whether thesit4⌬ that Sit4p phosphatase is required to activate Elongator function. Such activation might involve effects on either or zymocin-resistantsap⌬strains share any of the other
phenotypes shown by Elongator mutations. In addition the expression or the activity of the Elongator complex. However, when the mRNA levels ofTOT1/ELP1,TOT2/ to zymocin resistance, these phenotypes include slow
growth, temperature sensitivity, and hypersensitivity to ELP2,TOT3/ELP3,TOT4/KTI12, andTOT5were exam-ined by RT-PCR, essentially identical levels of mRNA 6-azauracil. As a control for these experiments we used
a strain deleted forTOT3/ELP3, which encodes a known for each of these Elongator components were found in wild-type and sit4⌬ strains (not shown). Furthermore, component of Elongator (Wittschieben et al. 1999;
Frohloff et al. 2001). Either loss ofSIT4 or deletion comparison of the protein levels of Tot1p to Tot5p in strains with or without high-copy SAP155 showed of multipleSAPgenes has already been shown to confer
a slow growth phenotype (Lukeet al.1996) that we have essentially identical protein levels (not shown). Thus, if Sit4p does affect Elongator function, it is more likely confirmed in our work (not shown). Bothsit4⌬and the
quadruplesapdeletion strain also conferred tempera- to result from post-translational effects. If Sit4p and Elongator do function in a common process, then a ture sensitivity and 6-azauracil hypersensitivity (Figure
7). Loss of Sap185p and Sap190p also conferred both double mutant would not be expected to show any addi-tional growth defect. Conversely, if they act in distinct phenotypes, although the level of 6-azauracil
hypersensi-tivity was lower and comparable to that shown by the processes, then the slow growth ofsit4⌬strains would be predicted to be additive with that of Elongator deletion mutations. We therefore crossed a sit4⌬strain with ei-ther TOT3/ELP3 or TOT4/KTI12 deletion strains in a common genetic background and compared the growth defect of the single and double knockout progeny. The absence of any additional growth defect whensit4⌬was combined with eithertot3⌬ortot4⌬(Figure 8) is consis-tent with the notion that the phosphatase and the Elon-gator complex may function in a common pathway.
Figure4.—High-copySAP155promotes zymocin resistance
inssd1-d2and SSD1-v1 strains. Tenfold serial dilutions (left to right) ofssd1-d2(AY925) orSSD1-v1(CY4029) strains with
DISCUSSION or without high-copySAP155 (pARB19) were spotted onto
YPD agar containing 65% (v/v) culture supernatant from the
GRX3and zymocin resistance:Glutaredoxins and thi-zymocin nonproducing K. lactis strain NK40 (control) and
oredoxins are small, heat-stable oxido-reductases con-onto YPD agar containing the indicated concentration of
vari-Figure6.—Loss ofSIT4or combined loss of SAP185and SAP190is resistant to intracellular zymocin expression. The indicated strains were transformed with either YCplac111 (as a control) or pLF16, which expresses the zymocin␥-subunit from theGALpromoter. Tenfold serial dilutions of cells were plated out on YP medium containing either 2% glucose or 2% galactose. While growth of thesap4⌬sap155⌬strain was inhibited by expression of the zymocin␥-subunit on galactose, strains lacking eitherSIT4or bothSAP185andSAP190were able to grow despite induction of theGAL-zymocin␥-subunit construct. The wild-type control strain (CY4029) behaved identically to CY5220 (not shown).
Figure5.—Sit4 phosphatase and eitherSAP185orSAP190
are required for zymocin sensitivity. Tenfold serial dilutions (left to right) of strains were spotted onto YPD agar (left) or
vatedGRX3dosage does not act by protecting the intra-YPD agar containing zymocin (middle) or assessed using the
eclipse assay (see Figure 3 legend). All strains were W303SSD1- cellular target from zymocin and that the most likely v1and either wild type (CY4029) or lackingSIT4(CY3938) or mode of action therefore concerns entry of zymocin one or moreSAPgenes as indicated (CY5220, CY5224, CY917,
into sensitive cells. Zymocin uptake is a process that is CY4380, and DJY8-DJY17; see Table 1). Note that the resistance
poorly understood at present, although initial binding ofsit4⌬and some multiplesap⌬strains is evident despite their
to sensitive cells requires the chitin binding domain and weaker growth.
chitinase activity of the␣-subunit (Butleret al.1991a;
Jablonowski et al.2001). Zymocin entry into the cell may involve the intensely hydrophobic -subunit, to ety of proposed roles, including protein folding and
which the zymocin ␥-subunit is known to be disulfide repair of oxidatively damaged proteins (Holmgren
bonded (Stark et al. 1990). If this disulfide bond is 1989). Grx3p and Grx4p are two highly related yeast
required for␥-subunit uptake, it is possible that higher-glutaredoxins that belong to a subfamily that also
in-than-normal extracellular levels of Grx3p might pro-cludes Grx5p. Unlike other glutaredoxins and
thioredox-mote reduction of this bond and thereby block toxin ins (including yeast Grx1p and Grx2p), Grx3p, Grx4p,
entry. Since it is most likely that Grx3p is not normally and Grx5p have only a single cysteine residue at the
an extracellular protein, it could be released by lysis of active site in place of the usual -C-X-X-C- motif (
Rodri-some proportion of cells in a colony, thereby protecting
guez-Manzaneque et al. 1999; Grant 2001). Similar
the remaining viable cells from zymocin. Alternatively, glutaredoxin-like proteins, apparently with a single,
ac-excess Grx3p within the cell might alter the normal tive-site cysteine, are also found in other eukaryotic
or-redox balance and interfere with release of the␥-subunit ganisms. Like protein disulfide isomerases (Freedmanet
in that way. Finally, high-copyGRX3might also function al.1994), which also contain thioredoxin-like domains,
less directly in blocking zymocin entry, for example, glutaredoxins can catalyze the reduction of disulfide
by leading to reduced cell-wall chitin levels. A better bonds using glutathione, although protein disulfide
understanding of the zymocin entry process will be re-duction is generally thought to involve a dithiol
mecha-quired to determine how GRX3 might antagonize zy-nism (Bushwelleret al.1992). It is therefore unclear
mocin function. whether Grx3p and its paralogues can function in this
The role of Sit4p phosphatase in zymocin sensitivity: way, although Grx3p and Grx4p do have an additional
Starting with the finding that high-copySAP155confers cysteine elsewhere in the polypeptide that might
con-resistance to K. lactis zymocin, we have demonstrated ceivably be involved in redox function. We have
identi-in this work that cells need a functional Sit4p proteidenti-in fiedGRX3as a sequence that protects cells fromK. lactic
phosphatase for zymocin sensitivity and that either zymocin in high copy, but even on the original pMA3a
Sap185p or Sap190p is also required. Although (like vector (which usesleu2dselection),
GRX3could not
pro-zymocin-treated cells)ssd1-d2 strains deficient in Sit4p tect cells from induction of the zymocin␥-subunit from
Figure7.—sit4⌬zymocin-resistant mutants share other phe-notypes with RNAPII Elongator mutants. In A, W303SSD1-v1 strains of the indicated genotype were tested for growth at 39⬚. Only the wild type andsap4⌬sap155⌬mutant show significant growth at this temperature, as indicated by the presence of individual colonies growing to the center of the plate. (B) Sensitivity of strains toward 6-azauracil. Tenfold serial dilutions of the indicated strains were plated on SD agar without uracil and either containing or lacking 50 g/ml 6-azauracil. All strains contained YCplac33 (carryingURA3).
Figure8.—Loss of Elongator function fails to show an
addi-tive defect with loss ofSIT4.W303SSD1-v1strains containing itself cannot be the intracellular target of the zymocin;
tot4/kti12⌬(LFY6; A) ortot3/elp3⌬(LFY5; B) mutations were SSD1-v1 strains in which Sit4p function is dispensable
crossed with CY4029 (sit4⌬) and sporulated, and tetrads were are sensitive to zymocin inhibition. Sap4p, Sap155p, dissected. The growth of spores from a typical tetrad from each Sap185p, and Sap190p are Sit4p-associated proteins that cross is shown, together with the doubling time (in minutes) could be either regulators or effectors of Sit4p function subsequently determined in liquid YPD medium.
and our data underscore the division of these four pro-teins into two families (Sap4p/155p and Sap185p/
190p), each of which has at least some unique function. Sap190p·Sit4p complexes. The alternative model, where-by either elevated dosage ofSAP155 or loss ofSAP185 Thus, only combined loss of Sap185p and Sap190p leads
to zymocin resistance, while combined loss of Sap4p andSAP190 promote zymocin resistance by increasing the amount of Sap155p·Sit4p in the cell, is inconsistent and Sap155p or triple sap deletion strains still
con-taining either Sap185p or Sap190p are zymocin sensi- with the finding that loss of Sit4p itself also causes resis-tance.
tive. This is consistent with previous work showing that
SAPgenes from one family are ineffective in high copy Another role of Sit4p phosphatase is in the TOR sig-naling pathway, where its interaction with Tap42p is at rescuing the growth defect shown by loss of the other
family and thatsap185⌬sap190⌬(but notsap4⌬sap155⌬) important (Di Como and Arndt 1996). Tap42p is bound to Sit4p and to the catalytic subunit of yeast PP2A is synthetically lethal with loss of BEM2, as is loss of
SIT4. (PP2AC) under conditions of nutrient sufficiency, but
on starvation or when the TOR kinases are inhibited High-copy SAP155 confers zymocin resistance by a
mechanism involving competition between the different by rapamycin, this interaction is lost. On release from Tap42p, Sit4p has been proposed to dephosphorylate SAPproteins, since its effect is antagonized by elevated
dosage of eitherSAP185 orSAP190.Although we have at least some of the downstream effectors of the TOR pathway (Beck andHall 1999). Since Tap42p binds not looked directly at the effect ofSAP155on the
associa-tion of the different SAPs with Sit4p, it has previously to Sit4p in a complex distinct from the Sap·Sit4p com-plexes we also tested whether high-copyTAP42 could been clearly demonstrated that high-copySAP155
effec-tively dissociates Sap185p and Sap190p from Sit4p im- confer zymocin resistance. However, its failure to do so suggests that it is unable to compete effectively with munoprecipitates, while, conversely, high-copySAP190
greatly reduced the level of Sap155p in Sit4p immune Sap185p and Sap190p for binding to Sit4p, despite evi-dence that more Sit4p can associate with Tap42p in sap-complexes (Luke et al. 1996). Thus, given that the
sap185⌬ sap190⌬ double mutant is zymocin resistant, deficient strains (Di Como and Arndt 1996). Since rapamycin also causes cells to arrest in G1 it is attrac-all our data are fully consistent with high-copySAP155
and L.F. by a Federation of European Microbiological Societies
Fellow-in blockFellow-ing growth-promotFellow-ing signalFellow-ing through the
ship. This work was supported by project grant FG94/518 from the
TOR pathway. Like rapamycin-treated cells, tap42-11
Agricultural and Food Research council to M.J.R.S. and by grants from
mutants arrest at their restrictive conditions because Deutsche Forschungsgemeinschaft (Scha 750/2-1 and 2-2) to R.S. they induce a range of responses that is normally
inhib-ited by TOR signaling; at their permissive temperature,
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