Creation of a fully active, cytosolic form of human type I 3-hydroxysteroid dehydrogenase/isomerase by the deletion of a membrane-spanning domain J L Thomas, B W Evans, G Blanco 1 , J I Mason 2 and R C Strickler 3 Department of Obstetrics and Gynecology, Washington University School of Medicine, St Louis, Missouri, USA 1 Department of Cell Biology and Physiology, Washington University School of Medicine, St Louis, Missouri, USA 2 Department of Reproductive and Developmental Sciences (Clinical Biochemistry), University of Edinburgh, Royal Infirmary of Edinburgh, Edinburgh, UK 3 Department of Obstetrics and Gynecology, Henry Ford Health System, Detroit, Michigan, USA (Requests for offprints should be addressed to J L Thomas, Department of Obstetrics and Gynecology, 4911 Barnes-Jewish Hospital Plaza, St Louis, Missouri 63110, USA) ABSTRACT Human 3-hydroxysteroid dehydrogenase/steroid  5 - 4 -isomerase (3-HSD/isomerase) is a bifunc- tional, single enzyme protein that is membrane- bound in the endoplasmic reticulum (microsomes) and mitochondria of cells in the placenta (type I) and in the adrenals and gonads (type II). Two membrane-binding domains (residues 72–89 and 283–310) have been predicted by analyses of hydrophobicity in the type I and II isoenzymes (90% regional homology). These putative membrane domains were deleted in the cDNA by PCR-based mutagenesis, and the two mutant enzymes were expressed by baculovirus in insect Sf9 cells. Differential centrifugation of the Sf 9 cell homoge- nate containing the 283–310 deletion mutant revealed that 94% of the 3-HSD and isomerase activities were in the cell cytosol, 6% of the activities were in the microsomes, and no activity was in the mitochondria. This is the opposite of the subcellular distribution of the wild-type enzyme with 94% of the activities in the microsomes and mitochondria and only 6% activity in the cytosol. The organelle distribution of the 72–89 deletion mutant lies between these two extremes with 72% of the enzyme activity in the cytosol and 28% in the microsomes/ mitochondria. The integrity of the subcellular organelle preparations was confirmed by electron microscopy. Western immunoblots confirmed the presence of the 283–310 deletion mutant enzyme and the absence of the wild-type enzyme in the insect cell cytosol. The unpurified, cytosolic 383–310 deletion mutant exhibited 3-HSD (22 nmol/min per mg) and isomerase (33 nmol/min per mg) specific activities that were comparable with those of the membrane-bound, wild-type enzyme. The isomerase reaction of the cytosolic 283–311 deletion mutant requires activation by NADH just like the isomerase of the microsomal or mitochondrial wild-type enzyme. In contrast, the 72–89 deletion mutant had low 3-HSD and isomerase specific activities that were only 12% of the wild-type levels. This innovative study identi- fies the 283–310 region as the critical membrane domain of 3-HSD/isomerase that can be deleted without compromising enzyme function. The shorter 72–89 region is also a membrane domain, but deletion of this NH 2 -terminal region markedly diminishes the enzyme activities. Purification of the active, cytosolic 283–310 deletion mutant will produce a valuable tool for crystallographic studies that may ultimately determine the tertiary/ quaternary structure of this key steroidogenic enzyme. Journal of Molecular Endocrinology (1999) 23, 231–239 INTRODUCTION Human placental 3-hydroxysteroid dehydrogenase (3-HSD, EC 1·1·1·145) and steroid  5 - 4 - isomerase (EC 5·3·3·1) catalyze the sequential con- version of 3-hydroxy-5-ene steroids (pregnenolone and dehydroepiandrosterone (DHEA)) to 3-oxo- 4-ene steroids (progesterone and androstenedione) 231 Journal of Molecular Endocrinology (1999) 23, 231–239 0952–5041/99/023–231  1999 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology.org