Figure 8.

dMPPE directly dephosphorylates α-Man-II. (A) Identification of phosphorylated proteins in dmppe mutants. Phosphorylated protein enrichment was performed as described in Materials and methods. Bands whose amount increased in the dmppe mutant sample were cut and identified by MS. Detailed results from the MS analysis are provided in Supplementary data. (B) Ectopic expression of α-Man-II in wild-type and dmppe mutant flies. (C) Identification of the phosphorylated peptide from the dmppe mutant. Myc-tagged α-Man-II was immunoprecipitated from wild-type and mutant flies. Trypsin-digested peptides were used for MS analysis. In the dmppe mutant sample, the m/z 1821.97 ion is lost with the appearance of an m/z 1901.94 ion. (D) dMPPE directly dephosphorylates α-Man-II in vitro. Phosphorylation and dephosphorylation reactions were performed as described in Materials and methods. Upper panel: Autoradiogram of SDS–PAGE of ³²P in vitro labelled GST-α-Man-II recombinant protein. Lower panel: The same gel blotted and probed with antibodies to GST. rMPPE represents purified recombinant dMPPE and rMPPE^Δ represents purified recombinant truncated dMPPE.