Localization of the long form of beta-1,4-galactosyltransferase to the plasma membrane and Golgi complex of 3T3 and F9 cells by immunofluorescence confocal microscopy.

Abstract

ABSTRACT1&1,4-Galactosyltransferase (GalTase) is localized to two subcellular compartments, the Golgi complex, where it participates in cellular glycosylation, and the plasma membrane, where it functions as a receptor for oligosaccharide ligands on opposing cells or in the extracellular matrix. The gene for GalTase encodes two nearly identical proteins that differ only in their N-terminal cytoplasmic domains: both short and long GalTases share an 11-aa cytoplasmic tail, but long GalTase has an additional 13-aa sequence on its cytoplasmic domain. In this study, we investigated the subcellular distribution of endogenous long GalTase in untransfected F9 and 3T3 cells by using confocal microscopy and antibodies specific for the 13-aa sequence unique to long GalTase. Long GalTase was found in the Golgi complex as expected; long GalTase was also found on the plasma membrane in cell-type-specific distributions. In 3T3 cells, long GalTase was evident on the basal surface of cells possessing a migratory phenotype, being concentrated at the leading and trailing edges; nonmigratory cells had little detectable surface immunoreactivity. In F9 cells, long GalTase was localized on the plasma membrane, being concentrated at the apical aspect of intercellular junctions. These results demonstrate that in 3T3 and F9 cells, long GalTase is present on the cell surface in addition to the Golgi complex. The pattern of surface expression shows cell-type specificity that is consistent with GalTase function in cellular interactions.

Mutational analysis of the cytoplasmic domain of β1,4-galactosyltransferase I: influence of phosphorylation on cell surface expression

“…GFP fluorescence was also detectable on the surface of specific transfectants in distinct patches on lamellipodia and filopodia (Fig. 5b), consistent with the previously described distribution of the endogenous GalT I (Youakim et al, 1994). The expression of GFP fluorescence on the cell surface was quantified for each transfectant; 38% of TL-GFP-expressing cells demonstrated GFP fluorescence on their surfaces, whereas only 13% of TS-GFP-expressing cells had any detectable surface expression (Fig.…”

“…The pellet was resuspended in 1 ml TEN. All fractions were subsequently resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by Western blotting using antibodies against the GalT I catalytic domain (Nguyen et al, 1994), the long GalT I cytoplasmic domain (Youakim et al, 1994), caveolin, GM130 and p115 (BD Transduction Laboratories).…”

“…The cell lysate was precleared by incubating 1 ml aliquots overnight at 4°C with 10 µl preimmune rabbit sera, followed by 1-2 hours with Protein A-Sepharose. The Protein A-Sepharose-bound IgG complexes were removed by centrifugation and the supernatants incubated at 4°C with Protein A-Sepharose beads precoated with either preimmune rabbit sera, rabbit sera specific for the GalT I catalytic domain (Nguyen et al, 1994) or the long cytoplasmic domain (Youakim et al, 1994). After 2-3 hours of incubation, the beads were collected by centrifugation, washed twice and resuspended in SDS-sample buffer for SDS-PAGE.…”

“…The synthesis of two GalT I polypeptides with distinct cytoplasmic domains suggested a mechanism to account for its dual subcellular distribution and function (Dinter and Berger, 1995;Lopez et al, 1991;Nilsson et al, 1991;Shur et al, 1998;Teasdale et al, 1992;Youakim et al, 1994). In this regard, previous results show that both GalT I isoforms can function biosynthetically in the Golgi complex.…”

Characterization of a Novel Ubiquitin-Conjugating Enzyme That Regulates β1,4-Galactosyltransferase-1 in Embryonic Stem Cells

“…Although both isoforms reside and function in the Golgi complex, only the long form of GalT1 behaves as a receptor on the cell surface in laminin-dependent intra-and intercellular adhesions. This characteristic of long GalT1 is due to the extra 13-amino-acid peptide sequence in the cytoplasmic NH 2 terminus [14,18,19]. In this study, we cloned and characterized a galactosyltransferase-associating protein (GTAP), using a yeast two-hybrid system to screen a murine embryonic library.…”