Fig 1.
PPT1 is palmitoylated by DHHC3 and DHHC7.
A) PPT1 was co-transfected with HA tagged DHHC enzymes to HEK293 cells. The cells were metabolically labeled with 17-ODYA and PPT1 was immunoprecipitated with anti-PPT1 antibodies. A fluorescent tag was introduced by click chemistry and samples were separated by SDS-PAGE. Fluorescent scanning of the gels detected that PPT1 is palmitoylated by DHHD3 and DHHC7 (top panel, boxed). The relative amount of immunoprecipitated PPT1 is shown (middle panel). The expression of the DHHC enzymes in the cell lysates was verified by immunoblotting with anti-HA antibodies (low panel). B) PPT1 was co-transfected with HA-DHHC3 followed by metabolic labeling, immunoprecipitation of PPT1 and click chemistry. One reaction was treated with hydroxylamine and one reaction with PBS. The addition of hydroxylamine eliminated the fluorescent signal of PPT1 (upper panel) demonstrating that the signal is specific for palmitoylation. Similar amounts of PPT1 were immunoprecipitated and expressed (middle panels). The level of HA-DHHC3 expression in the cell lysates was detected by anti-HA antibodies (lower panel). C) PPT1 is palmitoylated on cysteine residue 6 and can be detected in the cell media. Empty vector, PPT1 or PPT1 C6S were co-expressed with HA-DHHC3 in HEK293, followed by metabolic labeling with 17-ODYA, immunoprecipitation and click chemistry. The control treatment results in a low background fluorescent signal probably due to low levels of expression of endogenous PPT1. The expression and amount of immunoprecipitated PPT1 and PPT1 C6S are similar as detected by immunoblotting with anti-PPT1 antibodies (middle panels). The expression of HA-DHHC3 in was detected by immunoblotting with anti-HA antibodies (lower panel). D) The fluorescence signal intensity for PPT1 and PPT1 C6S were quantified and normalized relative to the amount of immunoprecipitated PPT1 using four independent repeats. The fluorescent signal of PPT1 signal is almost five fold higher than that observed with PPT1 C6S. E) Palmitoylated PPT1 can be detected in cells and media and the signal is reduced following hydroxylamine treatment. PPT1 was co-expressed with HA-DHHC3 in HEK293 cells followed by metabolic labeling both cells and media were collected and PPT1 was immunoprecipitated and labeled using click chemistry. The fluorescent signal of PPT1 palmitoylation is shown in the upper panel. This signal is eliminated by the addition of hydroxylamine. The amount of immunoprecipitated PPT1 and the level of expression in the lysates are shown in the middle panels and in both cell and media by immunoblotting with anti-PPT1 antibodies. The expression of HA-DHHC3 was detected by immunoblotting with anti-HA antibodies in the lower panel. E) Palmitoylation of endogenous PPT1 in human embryonic stem cells. WIBR3 wildtype and 6C, PPT1 knockout clone generated using CRISPR/Cas9 were metabolically labeled with 17-ODYA, followed by immunoprecipitation of PPT1 and click chemistry. We could distinguish fluorescent band appeared in the–HA treatment, that band could be eliminated by hydroxylamine (+HA). The band did not appear in negative control of immunoprecipitation with beads only (-mAB) or in the immunoprecipitation from the PPT1 knockout 6C clone. The lower panel confirms that PPT1 was immunoprecipitated using Western blot.
Fig 2.
PPT1 palmitoylation does not affect its subcellular localizations.
The localization of PPT1 and PPT1 C6S was examined by co-transfection with fluorescently tagged cellular markers. A-F) Colocalization of PPT1 (A-C) and PPT1 C6S (D-F) to the lysosome marker Lamp1. Both PPT1 and PPT1 C6S are partially localized to the lysosome compartment in COS7 cells. G-L) PPT1 and PPT1 C6S localized also to the Golgi as demonstrated by Golgi marker coexpresssion. M-R) Colocalization of PPT1 and PPT1 C6S to the endoplasmic reticulum is demonstrated using a fluorescent tag attached to the KDEL ER retention signal. The experiments were repeated three times and in each experiment at least 25 cells were imaged.
Fig 3.
PPT1 C6S exhibits elevated enzymatic activity in the cell lysates (A) and the cell media (C).
The activity of PPT1 in COS7 cells was monitored following the overexpression of either PPT1 or PPT1 C6S, or with the addition of either HA-DHHC3 or HA-DHHC3 dominant negative mutant. The activity of PPT1 was defined as 100%, and the activity in other treatments were calculated accordingly (N = 3). A) In the cell lysates, the addition of HA-DHHC3 reduced PPT1 activity to 81%, while the addition of the dominant negative HA-DHHC3 resulted in 92% activity. The expression of the PPT1 C6S mutant resulted in 142% activity, whereas the co-expression with HA-DHHC3 resulted in activity levels similar to that of the wildtype enzyme (103%). The addition of the HA-DHHC3 dominant negative enzyme elevated the activity to 118%. B) Western blots showing the relative expression of PPT1, HA-DHHC3, and alpha-tubulin (loading control). C) In the cell media the activity of either the single transfected PPT1 or PPT1 C6S differed significantly. However, this effect was eliminated following the co-expression with either active DHHC3 or with its dominant negative mutant. D) The expression of PPT1 in the media was examined by western blot analysis. In the presence of DHHC3 wildtype or dominant negative mutant there was a decrease in the amount of PPT1 in the media. **, p<0.01; ***, p<0.001.
Fig 4.
Michaelis-Menten graph of PPT1 and PPT1 C6S.
The Km and Vmax of PPT1 and PPT1 C6S were calculated using the Michaelis-Menten equation following plotting the results of enzymatic activity obtained with different concentrations of the substrate (N = 9) A) Michaelis-Menten graph derived from the cell lysates. B) Michaelis-Menten graph derived from the cell media.