Localization and ER membrane insertion of parathyroid hormone-related protein analyzed without effects of reporter...

Parathyroid hormone-related protein (PTHrP) is transported to both the secretory pathway and the nucleus/nucleolus by its dual targeting signals, that is, an N-terminal signal peptide and nuclear targeting signal. Curiously, reporter proteins such as enhanced green fluorescent protein strongly affect the localization of the fusion protein. Here, we report a novel methionine tag for ³⁵ S-labelling added to the C-terminus of its prepro-form, which has no methionine and cysteine residue other than the initiation methionine that enables analyses of the molecular mechanism of its dual localization without the effects of the reporter proteins. Mutational analyses including insertion of a glycosylation site for the tagged PTHrP revealed that the evolutionarily conserved regions in the signal peptide and the pro-region facilitate the redirection of ppPTHrP from the secretory pathway to the nuclear targeting pathway.