Mutational analysis of the Golgi retention signal of bovine beta-1,4-galactosyltransferase.
To examine the role of the NH2-terminal region of the 402-residue-long beta-1,4-galactosyltransferase (beta-1,4-GT), a series of mutants and chimeric cDNA were constructed by polymerase chain reaction and transiently expressed in COS-7 cells, the enzyme activities were measured, and the protein was localized in the cells by subcellular fractionation or indirect immunofluorescence microscopy. We showed earlier that the deletion of the amino-terminal cytoplasmic tail and transmembrane domain from GT abolishes the stable expression of this protein in mammalian cells (Masibay, A.S., Boeggeman, E., and Qasba, P.K. (1992) Mol. Biol. Rep. 16, 99-104). Further deletion analyses of the amino-terminal region show that the first 21 amino acids of beta-1,4-GT are not essential for the stable production of the protein and are consistently localized in the Golgi apparatus. In addition, analysis of hybrid constructs showed that residues 1-25 of alpha-1,3-galactosyltransferase can functionally replace the beta-1,4-GT amino-terminal domain (residues 1-43). This fusion protein also showed Golgi localization. On the other hand, the alpha-2,6-sialyltransferase/beta-1,4-GT fusion protein (alpha-2,6-ST/beta-1,4-GT) needed additional COOH-terminal sequences flanking the transmembrane domain of the alpha-2,6-ST for stability and Golgi localization. Substitution of Arg-24, Leu-25, Leu-26, and His-33 of the beta-1,4-GT transmembrane by Ile (pLFM) or substitution of Tyr by Ile at positions 40 and 41 coupled with the insertion of 4 Ile residues at position 43 (pLB) released the mutant proteins from the Golgi and was detected on the cell surface. Our results show that (a) the transmembrane domains of beta-1,4-GT, alpha-1,3-galactosyltransferase, and alpha-2,6-ST, along with its stem region, all play a role in Golgi targeting and participate in a common mechanism that allows the protein to be processed properly and not be degraded in vivo; (b) increasing the length of the transmembrane domain overrides the Golgi retention signal and directs the enzyme to the plasma membrane; and (c) the length of the hydrophobic region of the transmembrane domain of beta-1,4-GT is an important parameter but is not sufficient by itself for Golgi retention.
Laboratory of Mathematical Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
A S
AS
Masibay
P V
PV
Balaji
E E
EE
Boeggeman
P K
PK
Qasba
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D004274:DNA, Recombinant / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism*; D015151:Immunoblotting; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009237:N-Acetyllactosamine Synthase / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides; D011487:Protein Conformation; D021382:Protein Sorting Signals / Q000378:metabolism*; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism; D017384:Sequence Deletion; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D014162:Transfection
D004274:DNA, Recombinant; D009838:Oligodeoxyribonucleotides; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D009237:N-Acetyllactosamine Synthase; D000254:Sodium-Potassium-Exchanging ATPase