Arni S. Masibay, Petety V. Balaji, Elizabeth E. Boeggeman, and Pradman K. Qasba. Mutational Analysis of the Golgi Retention Signal of Bovine beta-1, 4-Galactosyltransferase. The Journal of Biological Chemistry Vol.268, No. 13, May 5, 1993, pp. 9908-9916.
To examine the role of the NH2-terminal region of the 402-residue-long beta-1, 4-galactosyltransferase ($-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 immunofluorescense 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 anaylse of the amino-terminal region show that the first 21 amino acids of beta-1, 4-GT are not essential 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-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 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 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.
