Sulfated biomolecules are omnipresent in biology. Highly diverse biomolecules are sulfated by an armada of sulfotransferases that depend on provision of active sulfate in the form of 3’-phospho-adenosine-5’-phosphosulfate (PAPS) for action. In humans, PAPS is exclusively provided by the two bifunctional PAPS synthases PAPSS1 and PAPSS2. A growing number of studies report patients with various mutations in the PAPSS2 gene with clinical phenotypes ranging from bone and cartilage mal-formations to severely disturbed androgen metabolism. PAPSS2 has been characterised as an unstable protein. The nucleotide APS (adenosine-5’-phosphosulfate) is a specific stabiliser of PAPSS2 in vitro. However, the cellular concentration of APS, and the related nucleotides PAPS and PAP are unknown. Within the cell, scaffold proteins or chaperones may be involved in stabilising the PAPSS2 protein. Furthermore, nuclear or cytoplasmic PAPSS variants were analysed within the context of DHEA sulfation. We observed that cytosolic PAPSS2 supported DHEA sulfation by cytoplasmic sulfotransferase SULT2A1 to a larger extent than cytosolic PAPSS1. PAPSS2 may hence be involved in various, yet unknown protein interactions. The present proposal combines the biochemical expertise of the Applicant with the profound endocrinology and translational knowledge of the Host as well as the excellent know-how for targeted metabolite quantification that exists in Birmingham. With these pillars we are in the unique position to correlate the cellular phenotypes with the different disease-causing PAPSS2 mutations, to find novel protein interaction partners of PAPS synthases by proteomics and to develop and apply innovative analytical methodology to quantify APS, PAPS and PAP within mammalian cells.