Endocrine Abstracts

Membrane proteins of the endoplasmic reticulum (ER) are involved in a wide array of essential cellular functions. Identification of the topology of membrane proteins can provide important insight into their mechanisms of action and biological roles. This is particularly important for membrane enzymes, since their topology determines the subcellular site where a biochemical reaction takes place and the dependence on luminal or cytosolic substrates and co-factor pools. The methods currently available for the determination of topology of proteins are rather laborious and require post-lysis or post-fixation manipulation of cells. In this work, we have developed a novel and simple method for defining intracellular localisation and topology of ER membrane proteins in living cells, based on the fusion of the respective protein with redox-sensitive green fluorescent protein (roGFP). We validated the method and demonstrated that roGFP fusion proteins constitute a reliable tool for the study of ER membrane protein topology, using as control microsomal 11β-hydroxysteroid dehydrogenase (11β-HSD) proteins whose topology has been resolved, and comparing with an independent approach. We then implemented this method to determine the membrane topology of six microsomal members of the 17β-HSD family. 17β-HSD enzymes catalyse the reduction/oxidation at the position 17 of the steroid backbone of androgens and oestrogens, thus playing a key role in the control of the balance of active and inactive sex steroids. Many 17β-HSDs have been implicated in different forms of sex-specific cancer and 17β-HSD inhibitors have attracted considerable interest as therapeutic targets. Appropriate access of such inhibitors to the respective intracellular compartment needs to be considered in drug development. Our results revealed a luminal orientation of the catalytic site for three 17β-HSD enzymes. Knowledge of the intracellular location of the catalytic site of these enzymes will enable future studies on their biological functions and roles in human diseases.

Disclosure: Swiss National Foundation.