Precursor sequence, processing, and urothelium-specific expression of a major 15-kDa protein subunit of asymmetric unit membrane.
The asymmetric unit membrane (AUM) is a highly specialized biomembrane elaborated by terminally differentiated urothelial cells. It contains quasi-crystalline arrays of 12-nm protein particles each of which is composed of six dumbbell-shaped subdomains. In this paper we describe the precursor sequence, processing and in vitro membrane insertion properties of bovine uroplakin II (UPII), a 15-kDa major protein component of AUM. The cDNA-deduced amino acid sequence revealed that UPII is synthesized as a precursor protein containing a cleavable signal peptide of approximately 26 amino acids, a long pro-sequence of approximately 59 residues harboring three potential N-glycosylation sites, and the mature polypeptide of 100 residues. In vitro translation of UPII mRNA demonstrated that UPII is indeed first synthesized as a 19-kDa precursor, which loses its signal peptide upon insertion into added microsomes; this process is accompanied by the acquisition of high mannose-type oligosaccharides giving rise to a 28-kDa precursor which is completely protected from the digestion by exogenous proteases. These results, together with the presence of a stretch of 25 hydrophobic amino acids at the C terminus, suggest that UPII protein is anchored to the lipid bilayer via its C-terminal membrane-spanning domain with its major N-terminal domain exposed luminally. The formation of the 15-kDa mature UPII requires the removal of the pro-sequence by a furin-like endoprotease. Since only mature UPII devoid of this pro-sequence can interact with 27-kDa uroplakin I, the proteolytic processing of UPII precursor may play an important role in regulating the assembly of AUM. Finally, we showed that genomic sequences cross-hybridizing with bovine UPII cDNA are present in many mammals suggesting that UPII performs a highly conserved function in the terminally differentiated cells of mammalian urinary bladder epithelium.
Lin
J H
JH
Ronald O. Perelman Department of Dermatology, Kaplan Comprehensive Cancer Center, New York University School of Medicine, New York 10016.
Wu
X R
XR
Kreibich
G
G
Sun
T T
TT
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D002417:Cattle; D002454:Cell Differentiation; D002458:Cell Fractionation; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism* / Q000648:ultrastructure; D002499:Centrifugation, Density Gradient; D003488:Cyanogen Bromide; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D017931:DNA Primers; D004847:Epithelial Cells; D004848:Epithelium / Q000378:metabolism; D015870:Gene Expression; D015723:Gene Library; D006801:Humans; D046911:Macromolecular Substances; D008322:Mammals; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D008861:Microsomes / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification; D011061:Poly A / Q000302:isolation & purification / Q000378:metabolism; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D012313:RNA / Q000302:isolation & purification / Q000378:metabolism; D012333:RNA, Messenger; D015183:Restriction Mapping; D013045:Species Specificity; D014162:Transfection; D014357:Trypsin; D001743:Urinary Bladder / Q000166:cytology / Q000378:metabolism*; D060170:Uroplakin II
D017931:DNA Primers; D046911:Macromolecular Substances; D008565:Membrane Proteins; D010446:Peptide Fragments; D012333:RNA, Messenger; C554658:UPK2 protein, human; D060170:Uroplakin II; D011061:Poly A; D012313:RNA; D004247:DNA; D014357:Trypsin; D003488:Cyanogen Bromide