TORC2-Dependent Ypk1-Mediated Phosphorylation of Lam2/Ltc4 Disrupts Its Association with the β-Propeller Protein Laf1 at Endoplasmic Reticulum-Plasma Membrane Contact Sites in the Yeast Saccharomyces cerevisiae
"> Figure 1
<p>(<b>A</b>) Laf1 co-localizes with Lam2 at ER-PM CS contact sites. A strain (yEPS23) co-expressing Laf1-mKate, GFP-Lam2, and Pil1-GFP, each from its respective endogenous chromosomal locus, was grown to mid-exponential phase and viewed directly under an epifluorescence microscope, as described in the Materials and Methods Section. (<b>B</b>) Localization of Lam2 and Laf1 to ER-PM CSs is reduced, but not abolished, in the <span class="html-italic">tether</span>∆ strain. A wild-type strain (YFR712) and an otherwise isogenic <span class="html-italic">tether</span>∆ strain (YFR704) expressing Lam2-mNG (left panels) and the wild-type strain (YFR729) and the otherwise isogenic <span class="html-italic">tether</span>∆ strain (YFR705) expressing Laf1-mNG (right panels) were grown to mid-exponential phase in YPD and viewed directly under an epifluorescence microscope, as described in the Materials and Methods Section. (<b>C</b>) Localization of Laf1 to ER-PM CSs requires Lam2 and Lam4. Upper panels, Strains expressing Laf1-mNG in a wild-type (YFR713), <span class="html-italic">lam2∆</span> (YFR714), <span class="html-italic">lam4∆</span> (YFR715), or <span class="html-italic">lam2∆ lam4∆</span> (YFR720) background, were grown to mid-exponential phase and viewed directly under an epifluorescence microscope. Lower panels, samples of equivalent amounts (as judged by the Pgk1 loading control) of extracts of the same cells shown in the upper panel were resolved by SDS-PAGE and analyzed by immunoblotting, as described in the Materials and Methods Section. (<b>D</b>) Localization of Dgr2 to ER-PM CSs requires Lam2 and Lam4. Upper panels, Strains expressing Dgr2-mKate in a wild-type (YFR657-B), <span class="html-italic">lam2∆</span> (YFR658-B), <span class="html-italic">lam4∆</span> (YFR659-B), or <span class="html-italic">lam2∆ lam4∆</span> (YFR650-B) background, were grown to mid-exponential phase and viewed directly under an epifluorescence microscope. Lower panels, samples of equivalent amounts (as judged by the Pgk1 loading control) of extracts of the same cells shown in the upper panel were resolved by SDS-PAGE and analyzed by immunoblotting, as described in the Materials and Methods Section. (<b>E</b>) Lam2 localizes to ER-PM CSs in the absence of Laf1 and Dgr2. A WT strain (YFR512-A) and an otherwise isogenic <span class="html-italic">laf1∆ dgr2∆</span> derivative (YFR613-A), each expressing GFP-Lam2, were grown to mid-exponential phase and viewed using an Elyra PS.1 structured illumination (SIM) fluorescence microscope.</p> "> Figure 2
<p>(<b>A</b>) Laf1 has a function in retrograde sterol transport. Serial 10-fold dilutions of WT GFP-Lam2 (YFR512-A), <span class="html-italic">laf1∆</span> GFP-Lam2 (YFR603), <span class="html-italic">dgr2∆</span> GFP-Lam2 (YFR607), and <span class="html-italic">laf1∆ dgr2∆</span> GFP-Lam2 (YFR613-A) were spotted on plates lacking (-) or containing AmB at the indicated concentrations. The plates were scanned after 2 days of growth at 30 °C. (<b>B</b>) Laf1 is more abundant than Dgr2. Extracts of cells expressing, as indicated, either Laf1-mKate (YFR635) or Dgr2-mKate (YFR657-A), each from their endogenous chromosomal locus, were resolved by SDS-PAGE and analyzed by immunoblotting, as described in the Materials and Methods Section. (<b>C</b>) Laf1 functions in the same retrograde sterol transport pathway as Lam2 and Lam4. Serial 10-fold dilutions of WT (BY4741), <span class="html-italic">lam2∆ lam4∆</span> (YFR513), <span class="html-italic">laf1∆</span> (<span class="html-italic">ymr102c∆</span>), and <span class="html-italic">lam2∆ lam4∆ laf1∆</span> (YFR734-A) were spotted on plates lacking (-) or containing AmB at the indicated concentrations. The plates were scanned after 2 days of growth at 30 °C.</p> "> Figure 3
<p>(<b>A</b>) Immobilized Laf1 and Dgr2 bind soluble fragments of Lam2 and Lam4 in vitro. Upper panel, structural motifs present in the Lam2(482–1259)-(His)<sub>6</sub> (pFR391) and Lam4(371–1177)-(His)<sub>6</sub> (pFR407) constructs. Lower panel, GST-Laf1 (pMT2), GST-Dgr2 (pFR404), and GST alone were expressed in <span class="html-italic">E. coli</span> from the vector pGEX4T-1 and purified. Samples were incubated in solution with, as indicated, either Lam2(482–1259)-(His)<sub>6</sub> or Lam4(371–1177)-(His)<sub>6</sub> for 1 h, and then the resulting complexes captured on glutathione-agarose beads, as described in the Materials and Methods Section. After washing, bound proteins were eluted, resolved by SDS-PAGE, and analyzed by immunoblotting. (<b>B</b>) The PH domain of Lam2 impedes its binding to Laf1. Upper panel, structural motifs present in the Lam2(482-1259)-(His)<sub>6</sub> (pFR391), Lam2(633-1259)-(His)<sub>6</sub> (pFR408), and Lam2(849-1259)-(His)<sub>6</sub> (pFR409) constructs. Lower panel, GST-Laf1 (pMT2) was purified from <span class="html-italic">E. coli</span>, incubated with the three different Lam2 fragments indicated for 1 h, then the resulting complexes were affinity-captured on glutathione-agarose beads, as described in the Materials and Methods Section. After washing, bound proteins were eluted, resolved by SDS-PAGE, and analyzed by immunoblotting. (<b>C</b>) The PH domain in Lam2 is dispensable for its interaction with Laf1. Upper panel, structural motifs present in the Lam2(482-1259)-(His)<sub>6</sub> (pFR391), Lam2(482-1259; T518A L519A)-(His)<sub>6</sub> (abbreviated AA) (pFR392), and Lam2(482-1259; ∆644-760-(His)<sub>6</sub> (abbreviated ∆PH) (pFR393) constructs. Lower panel, GST-Laf1 (pMT2) and GST-Laf1(S709A S710A) (abbreviated AA) (pFR396) were purified from <span class="html-italic">E. coli</span>, incubated with the three different Lam2 fragments indicated for 1 h, then the resulting complexes were affinity-captured on glutathione-agarose beads, as described in the Materials and Methods Section. After washing, bound proteins were eluted, resolved by SDS-PAGE, and analyzed by immunoblotting.</p> "> Figure 4
<p>(<b>A</b>) State of Lam2 phosphorylation does not affect Laf1 stability. Upper panel, the three documented Ypk1 phosphorylation sites in Lam2, and the hextuple “A” mutant (S44A T45A T518A L519A T1237A V1238A) and the hextuple “E” mutant (S44E T45E T518E L519E T1237E V1238E). Lower panel, extracts of <span class="html-italic">lam4∆</span> cells co-expressing Laf1-mKate with either GFP-Lam2<sup>WT</sup> (YFR728-A and YFR728-B), GFP-Lam2<sup>A</sup> (YFR721-A and YFR721-B), and GFP-Lam2<sup>E</sup> (YFR722-A and YFR722-B) were resolved by SDS-PAGE and analyzed by immunoblotting. (<b>B</b>) Phosphorylation state of Lam2 does not affect Laf1 retention at ER-PM CSs. The <span class="html-italic">lam4∆</span> strains co-expressing Laf1-mKate and, as indicated, WT GFP-Lam2 (YFR728-B), GFP-Lam2<sup>A</sup> (YFR721-B), or GFP-Lam2<sup>E</sup> (YFR722-B), were grown to mid-exponential phase in SCD-Trp and viewed under an epifluorescence microscope. (<b>C</b>) Stimulation of TORC2-dependent Ypk1 activation does not affect either Lam2 or Laf1 localization or level. Upper panels, <span class="html-italic">lam4∆</span> cells co-expressing WT GFP-Lam2 and Laf1-mKate (YFR633-A) cells were grown to mid-exponential phase, then either mock-treated (-) or treated (+Myr) with myriocin (1.25 µM) for 2 h before viewing under an epifluorescence microscope. Lower panels, extracts of another <span class="html-italic">lam4∆</span> strain co-expressing WT GFP-Lam2 and Laf1-mKate (YFR726-A) were prepared, treated with calf intestinal phosphatase, resolved by SDS-PAGE, and analyzed by immunoblotting. (<b>D</b>) Phospho-mimetic mutation in Lam2(482–1259)-(His)<sub>6</sub> reduce its affinity for GST-Laf1. Two Lam2(482–1259)-(His)<sub>6</sub> mutants, Lam2(482-1259; T518A L519A T1237A V1238A)-(His)<sub>6</sub> (pFR411) and Lam2(482–1259; T518E L519E T1237E V1238E)-(His)<sub>6</sub> (pFR413), were purified from <span class="html-italic">E. coli</span>, incubated with purified GST-Laf1 for 1 h, then the resulting complexes were affinity-captured on glutathione-agarose beads, as described in the Materials and Methods Section. After washing, bound proteins were eluted, resolved by SDS-PAGE, and analyzed by immunoblotting.</p> "> Figure 4 Cont.
<p>(<b>A</b>) State of Lam2 phosphorylation does not affect Laf1 stability. Upper panel, the three documented Ypk1 phosphorylation sites in Lam2, and the hextuple “A” mutant (S44A T45A T518A L519A T1237A V1238A) and the hextuple “E” mutant (S44E T45E T518E L519E T1237E V1238E). Lower panel, extracts of <span class="html-italic">lam4∆</span> cells co-expressing Laf1-mKate with either GFP-Lam2<sup>WT</sup> (YFR728-A and YFR728-B), GFP-Lam2<sup>A</sup> (YFR721-A and YFR721-B), and GFP-Lam2<sup>E</sup> (YFR722-A and YFR722-B) were resolved by SDS-PAGE and analyzed by immunoblotting. (<b>B</b>) Phosphorylation state of Lam2 does not affect Laf1 retention at ER-PM CSs. The <span class="html-italic">lam4∆</span> strains co-expressing Laf1-mKate and, as indicated, WT GFP-Lam2 (YFR728-B), GFP-Lam2<sup>A</sup> (YFR721-B), or GFP-Lam2<sup>E</sup> (YFR722-B), were grown to mid-exponential phase in SCD-Trp and viewed under an epifluorescence microscope. (<b>C</b>) Stimulation of TORC2-dependent Ypk1 activation does not affect either Lam2 or Laf1 localization or level. Upper panels, <span class="html-italic">lam4∆</span> cells co-expressing WT GFP-Lam2 and Laf1-mKate (YFR633-A) cells were grown to mid-exponential phase, then either mock-treated (-) or treated (+Myr) with myriocin (1.25 µM) for 2 h before viewing under an epifluorescence microscope. Lower panels, extracts of another <span class="html-italic">lam4∆</span> strain co-expressing WT GFP-Lam2 and Laf1-mKate (YFR726-A) were prepared, treated with calf intestinal phosphatase, resolved by SDS-PAGE, and analyzed by immunoblotting. (<b>D</b>) Phospho-mimetic mutation in Lam2(482–1259)-(His)<sub>6</sub> reduce its affinity for GST-Laf1. Two Lam2(482–1259)-(His)<sub>6</sub> mutants, Lam2(482-1259; T518A L519A T1237A V1238A)-(His)<sub>6</sub> (pFR411) and Lam2(482–1259; T518E L519E T1237E V1238E)-(His)<sub>6</sub> (pFR413), were purified from <span class="html-italic">E. coli</span>, incubated with purified GST-Laf1 for 1 h, then the resulting complexes were affinity-captured on glutathione-agarose beads, as described in the Materials and Methods Section. After washing, bound proteins were eluted, resolved by SDS-PAGE, and analyzed by immunoblotting.</p> "> Figure 5
<p>Phosphorylation of Lam2 blocks its interaction with Laf1. (<b>A</b>) GST-Lam2(482-1259)-(His)<sub>6</sub> (pFR391) purified from <span class="html-italic">E. coli</span> was incubated with Ypk1-(His)<sub>6</sub>-HA purified, as described in the Materials and Methods Section, from <span class="html-italic">S. cerevisiae</span> strain CGA84 containing plasmid BG1805 expressing Ypk1-(His)<sub>6</sub>-HA-3C-ZZ, in the absence (-) or presence (+) of excess Mg-ATP. The products were then resolved by PhosTag™ SDS-PAGE (8% acrylamide gel, 35 µM PhosTag reagent), and analyzed by immunoblotting. (<b>B</b>) Purified recombinant GST-Laf1 was incubated for 1 h with the unphosphorylated and phosphorylated Lam2(482–1259)-(His)<sub>6</sub>, prepared as shown in (A), for 1 h, then the resulting complexes were affinity-captured on glutathione-agarose beads. After washing, bound proteins were eluted, resolved by SDS-PAGE, and analyzed by immunoblotting.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Construction of Yeast Strains and Growth Conditions
2.2. Plasmids and Recombinant DNA Methods
2.3. Production and Purification of GST-Fusion Proteins
2.4. Production and Purification of Ypk1 from S. cerevisiae
2.5. Preparation of Ypk1-Phosphorylated Lam2 Fragment
2.6. In Vitro Binding Assay
2.7. Immunoblotting
2.8. Fluorescence Microscopy
3. Results
3.1. Localization of β-Propeller Proteins Laf1 and Dgr2 at ER-PM CSs Requires Lam2 and Lam4
3.2. Laf1 Is Required for Efficient Retrograde Removal of PM Ergosterol
3.3. Laf1 Physically Associates with Lam2 and the PH Domain of Lam2 Is Not Required for Its Interaction with Laf1
3.4. Ypk1-Mediated Phosphorylation of Lam2 Disrupts Its Association with GST-Laf1
4. Discussion
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Strain | Genotype | Source/Reference |
---|---|---|
BY4741 | MATahis3∆1 leu2∆0 met15∆0 ura3∆0 | Research Genetics, Inc. (Huntsville, AL, USA) |
BY4742 | MAT his3∆1 leu2∆0 lys2∆0 ura3∆0 | Research Genetics, Inc. |
YFR651-A | BY4741 Laf1-mKate::SpHIS5 | This study |
YFR652-B | BY4742 Laf1-mKate::SpHIS5 lam2∆::HygMX | This study |
YFR653-B | BY4742 Laf1-mKate::SpHIS5 lam4∆::KanMX | This study |
YFR654-B | BY4742 Laf1-mKate::SpHIS5 lam2∆::HygMX lam4∆:: KanMX | This study |
YFR660 | BY4741 Ypk1(L424A)::ura3- ypk2∆::KanMX Laf1-mKate::SpHIS5 | This study |
YFR657-A | BY4741 Dgr2-mKate:: SpHIS5 | This study |
YFR657-B | BY4742 Dgr2-mKate:: SpHIS5 | This study |
YFR658-B | BY4742 Dgr2-mKate:: SpHIS5 lam2∆::HygMX | This study |
YFR659-B | BY4742 Dgr2-mKate:: SpHIS5 lam4∆::KanMX | This study |
YFR650-B | BY4742 Dgr2-mKate:: SpHIS5 lam2∆::HygMX lam4∆:: KanMX | This study |
YFR512-A | BY4741 GFP-Lam2::URA3 | [37] |
YFR613-A | BY4742 GFP-Lam2::URA3 laf1∆::KanMX dgr2∆::KanMX | This study |
YFR513 | BY4741 lam2∆:: HygMX lam4∆::KanMX | [37] |
YFR633-A | BY4741 GFP-Lam2::URA3 Laf1-mKate::SpHIS5 | This study |
YFR726-A | BY4742 GFP-Lam2::URA3 Laf1-mKate::SpHIS5 | This study |
YFR635 | BY4742 Laf1-mKate::SpHIS5 | This study |
YFR728 | BY4742 Laf1-mKate GFP-Lam2::URA3 lam4∆::KanMX | This study |
YFR721 | BY4742 Laf1-mKate::SpHis5 GFP-Lam2(S44A T45A T518A L519A T1237A V1238A)::URA3 lam4∆::KanMX | This study |
YFR722 | BY4741 Laf1-mKate::SpHis5 GFP-Lam2(S44E T45E T518E L519E T1237E V1238E)::URA3 lam4∆::KanMX | This study |
yEPS23 | BY4741 GFP-Lam2::URA3 Laf1-mKate::SpHIS5 Pil1-BFP::KanMX | This study |
YFR713 | BY4741 Laf1-mNG::LEU2 | This study |
YFR714 | BY4741 Laf1-mNG::LEU2 lam2∆:: HygMX | This study |
YFR715 | BY4742 Laf1-mNG::LEU2 lam4∆:: KanMX | This study |
YFR720 | BY4742 Laf1-mNG::LEU2 lam2∆::HygMX lam4∆::KanMX | This study |
ymr102c∆ | BY4742 laf1∆::KanMX | Research Genetics, Inc. |
YFR734-A | laf1∆::KanMX lam2∆::HygMX lam4∆::KanMX | This study |
YFR603 | BY4741 GFP-Lam2::URA3 laf1∆::KanMX | This study |
YFR607 | BY4741 GFP-Lam2::URA3 dgr2∆::KanMX | This study |
CGA84 | MATaleu2∆1::GEV::NATMX pep4∆::HIS3 prb1∆1.6R ura3-52 trp1-1 lys2-801a leu2∆1 his3∆200 can1 GAL | [46] |
SEY6210 | MAT leu2-3,112 ura3-52 his3∆200 trp1∆901 suc2∆9 lys2-801a GAL | [47] |
YFR712 | SEY6210 Lam2-mNG::LEU2 | This study |
ANDY198 | SEY6210 ist2∆::hisMX6 scs2∆::TRP1 scs22∆::hisMX6 tcb1∆::kanMX6 tcb2∆::kanMX6 tcb3∆::hisMX6 | [48] |
YFR704 | ANDY198 Lam2-mNG::LEU2 | This study |
YFR729 | SEY6210 Laf1-mNG::LEU2 | This study |
YFR705 | ANDY198 Laf1-mNG::LEU2 | This study |
Plasmid | Description | Source/Reference |
---|---|---|
pGEX4T-1 | GST tag, bacterial expression vector | GE Healthcare, Inc. |
pFR398 | pGEX4T-1 Laf1(684–834) | This study |
pFR402 | pGEX4T-1 Laf1(684–834) S709S S710A | This study |
pFR399 | pGEX4T-1 Dgr2(1–128) | This study |
pFR403 | pGEX4T-1 Dgr2(1–128) S47A S48A | This study |
pFR203 | pGEX4T-1 Orm1(1–85) | [50] |
pMT2 | pGEX4T-1 Laf1 | This study |
pFR396 | pGEX4T-1 Laf1(S708A S709A) | This study |
pFR404 | pGEX4T-1 Dgr2 | This study |
pKM263 | GST tag, bacterial expression vector | |
pFR391 | pKM263-Lam2(482–1259)-His6 | This study |
pFR392 | pKM263-Lam2(482–1259) T518A T1237A-His6 | This study |
pFR393 | pKM263-Lam2(482–1259)∆PH(644–760)-His6 | This study |
pFR407 | pKM263-Lam4(371–1177)-His6 | This study |
pFR408 | pKM263-Lam2(633–1259)-His6 | This study |
pFR409 | pKM263-Lam2(849–1259)-His6 | This study |
pGEX6P-1 | GST tag, bacterial expression vector | GE Healthcare, Inc. |
pFR377 | pGEX6P-1 Lam2(482–1259) | This study |
pFR411 | pGEX6P-1 Lam2(482–1259) T518A L519A T1237A V1238A-His6 | This study |
pFR413 | pGEX6P-1 Lam2(482–1259) T518E L519E T1237E V1238E-His6 | This study |
BG1805 | C-terminal His6-HA-3C-ZZ tag, yeast expression vector | GE Healthcare, Inc. |
pJT4317 | BG1805 Ypk1-His6-HA-3C-ZZ | GE Healthcare, Inc. |
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Topolska, M.; Roelants, F.M.; Si, E.P.; Thorner, J. TORC2-Dependent Ypk1-Mediated Phosphorylation of Lam2/Ltc4 Disrupts Its Association with the β-Propeller Protein Laf1 at Endoplasmic Reticulum-Plasma Membrane Contact Sites in the Yeast Saccharomyces cerevisiae. Biomolecules 2020, 10, 1598. https://doi.org/10.3390/biom10121598
Topolska M, Roelants FM, Si EP, Thorner J. TORC2-Dependent Ypk1-Mediated Phosphorylation of Lam2/Ltc4 Disrupts Its Association with the β-Propeller Protein Laf1 at Endoplasmic Reticulum-Plasma Membrane Contact Sites in the Yeast Saccharomyces cerevisiae. Biomolecules. 2020; 10(12):1598. https://doi.org/10.3390/biom10121598
Chicago/Turabian StyleTopolska, Magdalena, Françoise M. Roelants, Edward P. Si, and Jeremy Thorner. 2020. "TORC2-Dependent Ypk1-Mediated Phosphorylation of Lam2/Ltc4 Disrupts Its Association with the β-Propeller Protein Laf1 at Endoplasmic Reticulum-Plasma Membrane Contact Sites in the Yeast Saccharomyces cerevisiae" Biomolecules 10, no. 12: 1598. https://doi.org/10.3390/biom10121598