Table 2.

Plasmid constructs used in this study

PlasmidDescriptionReference
pRS314 CEN6 TRP1 39
pRS316 CEN6 URA3 39
pRS314-PAN1Pan1p;BamHI/EcoRI fragment in pRS314 41
pRS316-PAN1Pan1p;BamHI/EcoRI fragment in pRS316 41
pRS316-HA-PAN1HA-Pan1p; HA-tagged PAN1 in pRS316 41
PRS314-PAN1-HAPCR was used to generate an AscI site immediately before the stop codon of Pan1p in pRS314-PAN1, and a cassette containing three copies of the HA epitope together with theADH1 terminator sequences was ligated to the AscI site
pGAL-HA-LR1HA-Pan1p(1–385) containing the first long repeat; generated by PCR and cloned in frame with the HA epitope underGAL1 promoter control in pRS316
pGAL-HA-LR2HA-Pan1p(386–846) containing the second long repeat; generated by PCR and cloned in frame with the HA epitope underGAL1 promoter control in pRS316
pRS4242 μmTRP1 8
pRS4252 μmLEU2 8
pRS424-END3End3p;XbaI/ClaI fragment in pRS424 41
pRS425-END3End3p;XbaI/ClaI fragment in pRS425 41
pGAL-END3 END3 was generated by PCR and placed under GAL1 promoter control in pRS314
pRS314-SLA1Sla1p; 4.3-kbXhoI/SacII fragment in pRS314
pRS316-SLA1Sla1p; 4.3-kb XhoI/SacII fragment in pRS316
pRS424-SLA1Sla1p; 4.3-kbXhoI/SacII fragment in pRS424
pRS424-SH3Sla1p(1–443) containing only the three SH3 domains. Sequence downstream of BglII in pRS424-SLA1 was removed (BglII/EcoRV) and replaced with theADH1 terminator of pGBT9 (BamHI/EcoRV).
pRS424-SLA1ΔSRSla1p(1–854) without the C-terminal Sla1p repeats. Sequence downstream of BamHI in pRS424-SLA1 was removed (BamHI/EcoRV) and replaced with theADH1 terminator of pGBT9 (BamHI/EcoRV)
pRS424-SRSla1p(856–1244) containing the C-terminal Sla1p repeats; generated by PCR and placed under SLA1 promoter control in pRS424
pGAL-SH3Sla1p(1–440) construct containing the three SH3 domains; generated by PCR and placed underGAL1 promoter control in pRS316
pGAL-SRSla1p(856–1244) construct containing the Sla1p repeats; generated by PCR and placed under GAL1 promoter control in pRS316
pGST-SH3GST-Sla1p SH3 domains (2–440); generated by PCR and cloned into pGEX-4T-1
pGST-SRGST-Sla1p repeats (856–1244); generated by PCR and cloned into pGEX-4T-1
pRS315-Myc-SLA1Myc-Sla1p. SLA1 open reading frame was generated by PCR and cloned in frame after three copies of the Myc epitope under SLA1 promoter control in pRS315
pGBT9Gal4(1–147) DNA-binding domain,TRP1 4
pGAD424Gal4(768–881) activation domain, LEU2 4
pPAN1-LR1Pan1p(98–385) construct containing the first long repeat in pGBT9; generated by PCR
pPAN1-LR2Pan1p(384–846) construct containing the second long repeat in pGAD424; identical to pPAN1.2 41
pPAN1-CTPan1p(902–1480) construct containing the C-terminal proline-rich region in pGBT9; 2.3-kb NcoI/SalI PAN1 fragment was cloned into SmaI/SalI sites of pGBT9
pEND3End3p(1–349) in pGBT9; generated by PCR
pEND3-EHEnd3p(1–114) construct containing the N-terminal EH domain in pGBT9; generated by PCR
pEND3-EREnd3p(254–349) construct containing the C-terminal repeats in pGBT9; identical to pEND3.4 41
pSLA1ΔSRSla1p(1–854) construct lacking the C-terminal Sla1p repeats in pGAD424. ABamHI site was first generated in front of the ATG codon in pRS424-SLA1ΔSR. SLA1, without the C-terminal repeats, was then removed by BamHI digestion and moved to pGAD424
pSLA1-SRSla1p(856–1244) construct containing only the C-terminal Sla1p repeats in pGAD424; generated by PCR
pSLA1-SRΔNPFSla1p(856–1238) construct containing the C-terminal Sla1p repeats without the NPF motif in pGAD424; generated by PCR