Somatic Cell and Molecular Genetics, Vol.21, No. 1, 1995, pp. 1-18 Analysis of Mutations in Alleles of the fur Gene from an Endoprotease-Deficient Chinese Hamster Ovary Cell Strain 1 Michael J. Spence, 2 Joseph F. Sucic, 2 Brian T. Foley, 2 and Thomas J. Moehring 1,2,3 2 Department of Microbiology and Molecular Genetics, 316 Stafford Hail, Markey Center for Molecular Genetics; and 3Vermont Cancer Center, The University of Vermont, Burlington, Vermont 05405 Received 21 October 1994 Abstract--RPE.40 mutant cells differ from wild-type Chinese hamster ovary (CHO-K1) cells in their increased resistance to Pseudomonas exotoxin A and their inability to process the insulin proreceptor and certain viral envelope proproteins. Northern analysis revealed that RPE. 40 cells maintained a substantially lower steady-state level of 4.0 kb fur mRNA than did CHO-K1 cells. Analysis of fur cDNAs showed that RPE.40 cells were diploid at the fur locus, and RPE.40 cells had a Cys (TGC) to Tyr (TAC) mutation in codon 196 of one allele (allele I). Approximately 25-30% of the CHO-K1 cells were also heterozygous (Tyr/Cys) at codon 196, and pre-mRNAs transcribed from the second allele (allele II) in RPE.40 cells were defectively spliced. All other pre-mRNAs were correctly spliced. Rapid turnover of defectively spliced transcripts may account for the reduced steady-state level of fur mRNA observed in RPE.40 cells. Our results provide a mechanistic basis for the endoprotease-deficient phenotype of RPE. 40 cells. INTRODUCTION Many proteins in eukaryotic cells are initially synthesized as large inactive precur- sors (1-8). These precursors must be pro- cessed to their active form by one or more cellular endoproteases. Prior to assembly of infectious particles, many pathogenic viruses require processing of their envelope propro- teins by a host cell enzyme (9-14). Cleavage of precursors for cellular polypeptides, viral envelope, proteins, and certain bacterial toxins typically occurs at specific paired or multiple basic amino acid residues (1-16). Kexin, an enzyme of the yeast Saccharo- myces cerevisiae, was the first endoprotease to be characterized (17, 18). Discovery of the yeast gene KEX2 led to identification of a transcription unit encoding a portion of a human endoprotease with structural similari- ties to kexin (19). This partial gene sequence was initially found immediately upstream of the FES/FPS oncogene (20). It has since been established (21-25) that this gene, designated fur, encodes the widely expressed furin endoprotease (also called PACE). Furin is a 90-kDa, membrane-associated, Ca2+-dependent, serine endoprotease that is predominantly localized in the trans-Golgi network (TGN) (16, 26, 27). From the TGN, furin is transported to and recycled from the cell surface (28). Mammalian furin protein domains have been deduced (29), including a subtilisn-like 1This workwas supported by National Institutes of Health Grant AI 09100 and the LucilleP. Markey Charitable Trust. 1 0740-7750/95/0100-0001507.50/0 9 1995 Plenum Publishing Corporation