Fig. 4.
DCFBA of TAPL-dependent peptide transport. (A and B) Labeled proteoliposomes (2.5 µg coreTAPL, 50 µg lipid) were incubated at 37 °C for 1 min (A) or 3 min (B) with 5 µM RRYCATTO488KSTEL and 3 mM Mg-ATP. DCFBA was performed with intensity traces for DiD-labeled liposomes (red) and luminal peptide RRYCATTO488KSTEL (blue). The relative amount of transported peptide was calculated from the ratio of fluorescence bursts in both channels above a certain offset (22 kHz for liposomes; 25 kHz for peptides). Only 25 s of the whole traces (5 min) are presented. Inset depicts single burst events of approximately 8 ms (arrows indicate the duration of the event). The laser power was kept constant: 488, 3 µW; 633, 1 µW. (C and D) Fitting the concentration distribution of transported RRYCATTO488KSTEL with a log Gaussian equation (black and yellow dashed lines) resulted in a mean internal peptide concentration Cav. (E) Accumulation of peptides in single liposomes. The actual number of transported peptides per liposome was quantified using standards as depicted in Fig. S6. A Gaussian fit of the transport rate distribution of liposomes (n = 51) results in a mean transport rate of 21 ± 9 peptides per liposome per minute. (F) Turnover number of TAPL. Proteoliposomes (50 µg lipid) containing coreTAPLmVenus (2.5 µg) were incubated with 10 µM RRYCATTO655KSTEL for 1 min at 37 °C in the presence of 3 mM Mg-ATP. The mean transport rate per TAPL complex was determined from individual liposomes (n = 47) containing one to three TAPL complexes. The transport rate distribution was fitted by a Gaussian equation resulting in a mean turnover number of 8 ± 2 peptides per minute. Laser powers were kept constant: 488, 4.5 µW; 633, 2 µW.