Fig. 2.
The translocation activity of the SpHtp1 is dependent on sulfate-modified cell-surface molecules. (A–J) Confocal microscopy images of RTG-2 cells: Cells incubated for 1 h with 15 μM mRFP(His)6 (A) did not show any mRFP fluorescence inside the cells in contrast to cells incubated with 3 μM SpHtp124-68mRFP(His)6 (B). The mutated full-length SpHtp1-mRFP fusion protein, in which the KRHLR amino acids had been substituted with GGHLG, did not show any translocation into the cells under identical conditions (C). The reported RxLR-protein translocation inhibitor inositol-1,4-diphosphate (19) did not affect the translocation of SpHtp124-68mRFP(His)6 (D). However, pretreatment of the cells for 48 h with 70 mM of the sulfotransferase inhibitor NaClO3 (30) strongly inhibited the translocation of this protein (E). A similar effect was observed when the cells were 3-h preincubated with 1.1 U of a type VI aryl-sulphatase from Aerobacter aerogenes (34) (F). Incubation of the cells for 1 h with 10 μg/mL of an antityrosine-phosphate specific antibody before the application of SpHtp124-68mRFP(His)6 only slightly reduced the uptake of this protein (G). A much stronger effect was observed when an anti-tyrosine-sulfate specific antibody was used under identical conditions (H). Using the modified tyrosine amino acid H-Tyr(PO3)-OH at a concentration of 5 mM to compete for the SpHtp124-68mRFP(His)6 binding, the fluorescence levels found inside the cells were not significantly affected compared with the control (I). In contrast, with the same amount of tyrosine-sulfate [H-Tyr(SO3)-OH] a much stronger inhibitory effect was observed (J). Red channel: mRFP fluorescence; white channel: differential interference contrast (viability controls can be found in SI Appendix, Fig. S4). (K) Flow cytometric quantification of the mRFP-fluorescence uptake into RTG-2 cells corresponding to the images shown in A–J. Cells were grown in 25-cm2 flasks and washed 5× with L15 medium before incubation with the respective protein concentration dilute in L15-medium containing 10% FCS. After 1-h incubation, the cells were washed 3× 5 min with PB), 1× 10 min with PBS containing TO-PRO-3 iodide in a 1:1,000 dilution, 2× 5 min with PBS adjusted to pH 5.5, and 2× 5 min with PBS adjusted to pH 8.5. Subsequently, the cells were detached with 1 mL of 0.5 mg/mL trypsin (+1 mM EDTA), resulting in samples with ∼2 × 106 cells/mL. From 10,000 events counted, a homogenous population of cell that were TO-PRO-3 iodide-negative was selected (on average ∼80% of all events) and further analyzed. Plotted are the MFI values averaged from three individual experiments. Error bars are the SD of the MFI values. Cells were incubated with 15 μM mRFP(His)6 (column 1), 3 μM SpHtp124-68mRFP(His)6 (column 2), 3 μM of the SpHtp124-198mRFP(His)6GGHLG mutant (column 3), or 3 μM SpHtp124-68mRFP(His)6 in combination with the indicated treatments (columns 5–10) or nontreated RTG-2 cells (column 11). Example raw data for one repeat can be found in the SI Appendix.