The transmembrane peptide DWORF activates SERCA2a via dual mechanisms

Figures

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  Figure 1. RFP–DWORF binding to GFP–SERCA2a in live cells.A, confocal microscopy images of GFP-SERCA (donor) and RFP–DWORF (acceptor) fluorescence signals. B, FRET efficiency E (1 − τD+A/τD) versus μg of acceptor DNA seeded per dish. C, fraction of SERCA2a containing bound DWORF (XDA). Error bars indicate SD (n = 3). GFP, green fluorescent protein; RFP, red fluorescent protein.

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  Figure 2. DWORF competes with PLB for SERCA binding.A, schematic illustration of the tested hypothesis, of competition between unlabeled DWORF and RFP-PLB for binding to GFP-SERCA2a. B, FRET efficiency E versus μg/well of DWORF DNA transfected into 6-well plates stably expressing GFP-SERCA2a and RFP-PLB. C, mole fraction (XDA) of GFP-SERCA (donor) bound to RFP-PLB (acceptor), for the same samples as in B. Error bars indicate SD (n = 3). GFP, green fluorescent protein; RFP, red fluorescent protein.

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  Figure 3. FRET-detected interaction of SERCA with the isolated TM domain of DWORF is similar to that with full-length DWORF.A, FRET efficiency E in HEK293 cells stably expressing GFP–SERCA2a, versus μg/dish of DNA transiently transfected into HEK293 cells expressing RFP-TM–DWORF (black) or full-length RFP–DWORF (red). B, fraction XDA of donors (GFP) transferring energy (bound) to acceptor (RFP) determined for samples in panel A. C, FRET efficiency E for increasing amounts of TM–DWORF DNA transfected into a stable cell line expressing GFP–SERCA2a and RFP–PLB. D, fraction XDA of donors transferring energy (bound) to acceptors determined for samples in C. Error bars indicate SD (n = 3). GFP, green fluorescent protein; RFP, red fluorescent protein.

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  Figure 4. P15 and W22 residues of DWORF (human isoform) are essential for the full extent of DWORF–SERCA2a complex formation.A, DWORF amino acid sequence alignment (19). Blue region denotes the putative TM domain. ∗ indicates a residue conserved among all species shown. B, helical wheel of DWORF TM domain, with P15 and W22 indicated by red circles. C, FRET efficiency of RFP–DWORF single-mutant constructs expressed with GFP-SERCA2a. In total, 5 μg/well of each mutant DWORF DNA was transfected into a stable cell line expressing GFP–SERCA2a. D, FRET efficiency (black; FLT measurement excited at 481 nm) and RFP fluorescence intensity (blue; excited at 532 nm) of RFP–DWORF double-mutant constructs expressed under similar conditions. In total, 5 μg/well of each double-mutant DWORF DNA was transfected into a stable cell line expressing GFP–SERCA2a. E, fraction XDA of donors transferring energy (bound) to acceptor determined for samples in D. F, FRET efficiency E of RFP-PLB to GFP-SERCA2a versus μg/well DNA of untagged-DWORF (P15A/W22A) transfected. Error bars indicate SD (n = 3). GFP, green fluorescent protein; RFP, red fluorescent protein.

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  Figure 5. DWORF activates SERCA2a in the absence of PLB.A, Ca-ATPase activity measured in homogenates of cells expressing GFP-SERCA2a and unlabeled DWORF (black, pKCa = 6.36 ± 0.04, Hill n = 0.82 ± 0.02), GFP-SERCA2a only (red, pKCa = 6.08 ± 0.03, Hill n = 1.26 ± 0.03), or GFP–SERCA2a pretreated with 100 nM thapsigargin (Tg) (blue). B, Ca-ATPase activity measured in homogenates of cells expressing GFP-SERCA2a and unlabeled DWORF–P15/W22 (black, pKCa = 6.34 ± 0.04, Hill n = 1.28 ± 0.01), GFP-SERCA2a only (red, pKCa = 6.32 ± 0.04, Hill n = 1.13 ± 0.02), or GFP-SERCA2a pretreated with 100 nM thapsigargin (Tg) (blue). GFP-SERCA2a was stably expressed in HEK293 cells, then 20 μg of DNA per dish (for unlabeled DWORF or unlabeled mutant DWORF) was transiently transfected into the cells. Ca-ATPase assays were performed using cell homogenates, as described in Experimental procedures. Data were fitted using V = V0 + Vmax/(1 + 10−n[pKCa − pCa]), to determine pKCa, the pCa value for half-maximal activation by Ca. PLB causes a decrease in pKCa (increase in [Ca2+] required for SERCA activation), so activation of SERCA corresponds to an increase in pKCa. Activity was normalized to the control (SERCA2a…