TABLE 2.

Plasmids used in this study

PlasmidDescriptionSource/reference
pZL473pRS416 (CEN URA3) containing PTR3This study
pZL1179pRS416 containing PTR3-myc; expresses Ptr3 with a 6× myc tag inserted between Ptr3 residues 157 and 158This study
pEG-KG-YCK12 μm URA3 Leu2-d plasmid containing GAL1-GST-YCK136
pEG-KG-YCK22 μm URA3 Leu2-d plasmid containing GAL1-GST-YCK236
pZL1834pRS416 containing STP1-HA; expresses Stp1 with 3× HA tagged at the C-terminal endThis study
pZL1949pRS416 containing HIS6-PTR3-myc; expresses Ptr3 with a six-His tag at the N-terminal end and a 9× myc tag inserted between residues 157 and 158 of Ptr3This study
pZL2107pRS416 containing HIS6-PTR3(S321A)-mycThis study
pZL2112pRS416 containing HIS6-PTR3(T635A)-mycThis study
pZL2122pRS416 containing HIS6-PTR3(T525A)-mycThis study
pZL2125pRS416 containing HIS6-PTR3(T525D)-mycThis study
pZL2132pRS416 containing HIS6-PTR3(T525E)-mycThis study
pZL2323Yip352 containing PTR3(Q439R)-myc
pZL736pRS416 containing SSY5This study
pZL1668pRS416 containing SSY5-HA; expresses Ssy5 with a 3× HA tag at the C-terminal endThis study
pZL1239pRS417 (CEN LYS2) containing PTR3-myc; expresses Ptr3 with a 9× myc tag inserted between residues 157 and 158 of Ptr3This study
pZL835pRS416 containing PTR3-HA; expresses Ptr3 with a 3× HA tag inserted between Ptr3 residues 157 and 158This study
pZL840pRS416 containing HA-SSY5; expresses Ssy5 with a 6× HA tag at the N-terminal end of Ssy5aThis study
pZL465pWCJ (CEN URA3) containing AGP1-lacZThis study
pZL2230pRS417 containing STP1-HA; expresses Stp1p with 3× HA tagged at the C-terminal endThis study
pGBKT7A yeast two-hybrid plasmid carrying Gal4 DNA binding domain encoding sequenceClontech Laboratories, Inc.
pGBKT7-Lst8pGBKT7 containing the LST8 open reading frame (ORF)This study
pGBKT7-SSY1NpGBKT7 carrying DNA sequence encoding amino acid residues 1-284 of Ssy1This study
pACTIIA yeast two-hybrid plasmid carrying Gal4 DNA binding domain encoding sequenceClontech Laboratories, Inc.
pACTII-LST8pACTII carrying the LST8 ORFThis study
pACTII-PTR3pACTII carrying the PTR3 ORFThis study
pACTII-SSY5pACTII carrying the SSY5 ORFThis study
pACTII-YCK1pACTII carrying the YCK1 ORFThis study
  • a The primer pair gtcaACTAGTGTCACGGCGAATCGATCTAT and gtcaACGCGTCATCGGTATATCGAGTTTAC (lowercase letters indicate nucleotides introduced to facilitate digestion of PCR products by restriction endonucleases) was used to amplify a 1,003-bp DNA sequence containing the SSY5 promoter region plus the start codon by PCR. The primer pair gtcaACGCGTGTCAGATTTTTTGGTTTAAACAAGAAAAAGAACGA and gtcaGTCGACCTGTGAACCAAGGTACCTTC was used to amplify the SSY5 ORF plus a 376-bp 3′ untranslated region sequence. These two DNA fragments were digested with SpeI and MluI and with MluI and SalI, respectively, and cloned into SpeI- and SalI-digested pRS416 to form pZL737, thus introducing an MluI site right after the start codon of SSY5. Two tandem repeats of HA3 flanked by MluI sites were cloned into the MluI site of pZL737 to form pZL840.