Abstract
To investigate the organization of Golgi glycosyltransferases and their mechanism of localization, we have compared the properties of a number of medial and late acting Golgi enzymes. The medial Golgi enzymes, N-acetylglucosaminyltransferase I and II (GnTI and GnTII) required high salt for solubilization and migrated as high molecular weight complexes on sucrose density gradients. In contrast, the late acting Golgi enzymes, beta1,4-galactosyltransferase and alpha1, 2-fucosyltransferase, were readily solubilized in low salt and migrated as monomers/dimers by sucrose density gradient centrifugation. Analysis of membrane-bound GnTI chimeras indicates that the formation of high molecular weight complexes does not require the transmembrane domain and cytoplasmic tail sequences of GnTI. Furthermore, a soluble form of GnTI, containing the stem region and catalytic domain, accumulated in the Golgi prior to secretion, in contrast to beta1,4-galactosyltransferase. Soluble GnTI, which also associated with high molecular weight complexes, was comparable with membrane-bound GnTI in its ability to glycosylate newly synthesized glycoproteins in vivo. Mutation of charged residues within the stem region of GnTI, known to be important for "kin recognition", had no effect on the efficiency of Golgi localization, the inclusion into high molecular weight complexes, nor functional activity in vivo. The differences in behavior between the medial and late acting Golgi enzymes may contribute to their differential localization and their ability to glycosylate efficiently in the correct Golgi subcompartment.
Highlights
To investigate the organization of Golgi glycosyltransferases and their mechanism of localization, we have compared the properties of a number of medial and late acting Golgi enzymes
All Golgi glycosyltransferases cloned to date are Nin/Cout membrane proteins containing a short N-terminal cytoplasmic domain, a single hydrophobic membrane-spanning domain, and a large carboxyl-terminal catalytic domain situated in the lumen of the Golgi apparatus (4 – 6)
We have shown that inclusion of the medial Golgi enzyme, N-acetylglucosaminyltransferase I (GnTI), in high molecular weight complexes does not require the transmembrane domain of GnTI but rather is dependent on the luminal domain
Summary
(Received for publication, August 17, 1999, and in revised form, December 23, 1999). From the Department of Pathology and Immunology, Monash University Medical School, Melbourne, Victoria 3181, Australia. Soluble GnTI, which associated with high molecular weight complexes, was comparable with membranebound GnTI in its ability to glycosylate newly synthesized glycoproteins in vivo. Analysis of glycosyltransferase chimeras from transfected eukaryotic cells has demonstrated that the transmembrane domain of glycosyltransferases plays a critical role in Golgi localization; in addition, in a number of cases contributions from the luminal domain and cytoplasmic tail have been detected, suggesting that there may be multiple signals involved in the specific localization of these Golgi enzymes We propose that the inclusion of GnTI into high molecular weight complexes defines the location of the soluble construct and is critical for its ability to coordinate the synthesis of complex N-glycans
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
