Förster transfer recovery reveals that phospholamban exchanges slowly from pentamers but rapidly from the SERCA regulatory complex

Abstract

Phospholamban (PLB) or the sarcoplasmic reticulum Ca2+-ATPase (SERCA) were fused to cyan fluorescent protein (CFP) and coexpressed with PLB fused to yellow fluorescent protein (YFP). The expressed fluorescently tagged proteins were imaged using epifluorescence and total internal reflection fluorescence microscopy. YFP fluorescence was selectively bleached by a focused laser beam. CFP fluorescence at the targeted site increased after YFP photobleaching, indicating fluorescence resonance energy transfer between CFP-SERCA/CFP-PLB and YFP-PLB. The increased donor fluorescence relaxed back toward baseline as a result of donor diffusion and exchange of bleached YFP-PLB for unbleached YFP-PLB, which restored fluorescence resonance energy transfer. Requenching of CFP donors, termed Förster transfer recovery (FTR), was quantified as an index of the rate of PLB subunit exchange from the PLB:SERCA and PLB:PLB membrane complexes. PLB subunit exchange from the PLB:SERCA regulatory complex was rapid, showing diffusion-limited FTR (tau=1.4 second). Conversely, PLB:PLB oligomeric complexes were found to be stable on a much longer time scale. Despite free lateral diffusion in the membrane, they showed no FTR over 80 seconds. Mutation of PLB position 40 from isoleucine to alanine (I40A-PLB) did not abolish PLB:PLB energy transfer, but destabilization of the PLB:PLB complex was apparent from an increased FTR rate (tau=8.4 seconds). Oligomers of I40A-PLB were stabilized by oxidative crosslinking of transmembrane cysteines with diamide. We conclude that PLB exchanges rapidly from its regulatory complex with the SERCA pump, whereas subunit exchange from the PLB oligomeric complex is slow and does not occur on the time scale of the cardiac cycle.

Figure 1

Left, TIRF microscopy of AAV-293 cells showed CFP-SERCA in the ER. Middle, YFP-PLB was localized to both the PM and ER. Right, Overlay. Areas of colocalization of CFP-SERCA and YFP-PLB appear white. Scale bar=5 μm.

Figure 2

Left, Pre- and postbleach epifluorescence images of CFP-SERCA and YFP-PLB expressed in AAV-293 cells. Laser spot photobleaching (514 nm) selectively ablated YFP at the target site (arrows), abolishing CFP-YFP energy transfer and increasing CFP fluorescence. Right, Ratio image (CFP-SERCA postbleach/prebleach) showing a spatially resolved 20% increase in CFP fluorescence after YFP photobleaching. Scale bar=μm.

Figure 3

A, Profiles of CFP-SERCA (blue circles) and YFP-PLB (green triangles) fluorescence at 2.5 seconds after YFP-selective laser spot photobleaching. The data are well described by Gaussian fits. B, As in A, plotted to show the relationship between CFP-SERCA and YFP-PLB fluorescence across the target region. The y-intercept indicates 13% energy transfer. C, Average YFP-PLB fluorescence (green triangles) decreased after acceptor-selective laser spot photobleaching and then recovered. CFP-SERCA fluorescence (blue circles) increased after acceptor photobleaching and then relaxed exponentially. D, FTR indicates regulatory complex subunit exchange with a time constant of 1.4 seconds. AU indicates arbitrary units.

Figure 4

A, Spatial profiles of CFP-PLB (blue circles) and YFP-PLB (green triangles) fluorescence at 2.5 seconds after YFP-selective laser spot photobleaching. The data are well described by Gaussian fits. B, As in A, plotted to show the relationship between CFP-PLB and YFP-PLB fluorescence across the target region. Y-intercept indicates 54% energy transfer. C, Average YFP-PLB fluorescence (green triangles) decreased after acceptor-selective laser spot photobleaching and then recovered. CFP-PLB fluorescence (blue circles) increased after acceptor photobleaching, then relaxed exponentially. D, Wild-type PLB (black circles) line-out Gaussian volume remained high over 80 seconds, indicating that the wtPLB pentamer did not undergo subunit exchange. PLB mutation I40A did not abolish FRET but decreased oligomeric stability, resulting in subunit exchange with a time constant of 8.4 seconds (red triangles). I40A oligomer stability was restored by crosslinking with diamide (blue squares). AU indicates arbitrary units.

Figure 5

Immunoblot of YFP fusions of PLB variants probed with anti-PLB monoclonal antibody 2D12 showing pentamer (P) and monomer (M) forms. Lanes from left are: wtPLB (1), oligomer-destabilized PLB (I40A-PLB) (2), I40A+diamide (3), I40A-PLB +Diamide +βME (4), Cys-null PLB (AFA-PLB) (5), AFA-PLB+diamide (6), and untransfected AAV-293 cell homogenate control (7). The intermediate-sized band (*) present in all samples is a non-PLB cross-reaction.

Figure 6

F/F₀ ratio images of CFP fusion proteins after YFP-selective spot photobleaching. Scale bar=10 μm.

Figure 7

Model of PLB subunit (PLB₁) exchange from pentamers (PLB₅) and the regulatory complex (PLB:SERCA). PLB associates rapidly with the SERCA pump and exchanges within seconds from the regulatory binding site. PLB exchange from pentamers is much slower, and oligomeric interactions are stable over many tens of seconds.