Nucleotide sequence of rat preputial gland beta-glucuronidase cDNA and in vitro insertion of its encoded polypeptide into microsomal membranes.
We have selected the rat preputial gland beta-glucuronidase as a model protein to study the sorting of newly synthesized lysosomal hydrolases to the lysosome. The complete coding sequence of beta-glucuronidase messenger RNA was determined from the sequences of a group of overlapping cDNA clones isolated from preputial gland cDNA libraries. The beta-glucuronidase mRNA primary translation product contains 648 amino acids, including an amino-terminal signal sequence of 22 residues. The polypeptide has four potential sites for the addition of asparagine-linked core oligosaccharides. A 376-residue segment of beta-glucuronidase shows extensive homology (23% sequence identity) to a portion of Escherichia coli beta-galactosidase. This homology most likely reflects an evolutionary relationship between the bacterial and eukaryotic enzymes and the conservation of structural features necessary for the glycosidase activity of both proteins. Translation of mRNA synthesized in vitro by transcription of a cDNA containing the entire beta-glucuronidase coding region yielded a polypeptide that was immunoprecipitated with anti-beta-glucuronidase antiserum and had the same electrophoretic mobility as the primary translation product of natural beta-glucuronidase mRNA. In the presence of microsomal membranes, the in vitro-synthesized beta-glucuronidase underwent cotranslational incorporation into the microsomes, as indicated by removal of the signal sequence and the addition of several oligosaccharide chains. The beta-glucuronidase cDNA will provide a useful tool to study the mechanism of mannose phosphorylation and other aspects of the sorting of lysosomal enzymes to lysosomes.
Nishimura
Y
Y
Rosenfeld
M G
MG
Kreibich
G
G
Gubler
U
U
Sabatini
D D
DD
Adesnik
M
M
Andy
R
R
D000818:Animals; D002474:Cell-Free System; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D005786:Gene Expression Regulation; D005837:Genitalia, Male / Q000201:enzymology; D005966:Glucuronidase / Q000235:genetics* / Q000378:metabolism; D006031:Glycosylation; D008297:Male; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008861:Microsomes / Q000201:enzymology; D008970:Molecular Weight; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000378:metabolism; D051381:Rats
D008565:Membrane Proteins; D021382:Protein Sorting Signals; D004247:DNA; D005966:Glucuronidase