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 trans iently expressed in COS-7 cells, the enzyme activities were measured, and the protein was localized in the cells by subcellular fractionatio n or indirect immunofluorescence microscopy. We showed earlier that th e deletion of the amino-terminal cytoplasmic tail and transmembrane do main from GT abolishes the stable expression of this protein in mammal ian cells (Masibay, A. S., Boeggeman, E., and Qasba, P. K. (1992) Mol. Biol. Rep. 16, 99-104). Further deletion analyses of the amino-termin al region show that the first 21 amino acids of beta-1,4-GT are not es sential for the stable production of the protein and are consistently localized in the Golgi apparatus. In addition, analysis of hybrid cons tructs showed that residues 1-25 of alpha-1,3-galactosyltransferase ca n functionally replace the beta-1,4-GT amino-terminal domain (residues 1-43). This fusion protein also showed Golgi localization. On the oth er hand, the alpha-2,6-sialyltransferase/beta-1,4-GT fusion protein (a lpha-2,6-ST/beta-1,4-GT) needed additional COOH-terminal sequences fla nking the transmembrane domain of the alpha-2,6-ST for stability and G olgi 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 resi dues at position 43 (pLB) released the mutant proteins from the Golgi and was detected on the cell surface. Our results show that (a) the tr ansmembrane domains of beta-1,4-GT, alpha-1,3-galactosyltransferase, a nd 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 protei n 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 leng th of the hydrophobic region of the transmembrane domain of beta-1,4-G T is an important parameter but is not sufficient by itself for Golgi retention.