In agreement with this idea, in the inactivation-removed mutant, the fluorescence upward deflection is clearly reduced at all potentials studied (Fig. 7), meaning that, during depolarizations, TMRM attached at E519C was less able to detect environmental changes associated with pore opening in S620T.
We found that in this mutant the fluorescence has a completely different distribution and accumulates in discrete vesicles (Fig. 4).
Concentration response and inhibition curves were obtained for each mutant, and the fluorescence values were normalized to the maximal response.
In the sur2Δ mutant cells, the fluorescence loss in the bud was significantly faster than in wild type cells (BI = 12 ± 1.4 for Nup49, Figure 4A and 4.9 for Nsg1, Figure 4B).
In adg1-1/tpt-2, adg1-1/tpt-2/pgm1-1, tpt-2/pgm1 and mex1-2/tpt-2, Fm was severely diminished compared to the wild type or the sex1-3/tpt-2 double mutant, whereas the fluorescence of free antennae was enhanced indicating that a severe restriction in leaf carbohydrate metabolism promotes a massive loss of PSII integrity under HL-conditions.
For the W7F/L135C mutant, the W14 fluorescence is strongly quenched in the Ib intermediate, showing that the non-native interactions observed in the equilibrium molten globule are also present in the kinetic Ib intermediate.
Likewise, the Ub-Fur4-GFP fusion protein was also efficiently cleared from the plasma membrane in the ubp2 deletion mutants, as the fluorescence signal detected at the plasma membrane was virtually absent one hour after uracil induction.
For endpoint measurements, the FRET substrate was mixed with 270 µM wild type or mutant Spy0129 and the fluorescence emission at 410 nm was measured before (T = 0) and after incubating the reaction for 3 hours (T = 3 hr) at 37°C.
Intriguingly, in cells expressing the S75A, S90A, and the S75A/S90D mutants the cortical fluorescence for F-actin and insulin seemed to be reduced compared to INS-1 and GFP-β2-syntrophin INS-1 cells (Fig. S7 and not shown).
However, in leaves of As-treated mother plants and two mutants, the red fluorescence was detected in trifoliate leaves with more severe effects on mother plants.
In contrast, mutant M43 with half the fluorescence intensity of wild type phaC had about 10% of the normal PHB accumulation found in wild type, showing a lower detection limit.
