Localization of three human polypeptide GalNAc-transferases in HeLa cells suggests initiation of O-linked glycosylation throughout the Golgi apparatus.
O-glycosylation of proteins is initiated by a family of UDP-N-acetylgalactosamine:polypeptide N-acetylgalactos-aminyltransferases (GalNAc-T). In this study, we have localized endogenous and epitope-tagged human GalNAc-T1, -T2 and -T3 to the Golgi apparatus in HeLa cells by subcellular fractionation, immunofluorescence and immunoelectron microscopy. We show that all three GalNAc-transferases are concentrated about tenfold in Golgi stacks over Golgi associated tubular-vesicular membrane structures. Surprisingly, we find that GalNAc-T1, -T2 and -T3 are present throughout the Golgi stack suggesting that initiation of O-glycosylation may not be restricted to the cis Golgi, but occur at multiple sites within the Golgi apparatus. GalNAc-T1 distributes evenly across the Golgi stack whereas GalNAc-T2 and -T3 reside preferentially on the trans side and in the medial part of the Golgi stack, respectively. Moreover, we have investigated the possibility of O-glycan initiation in pre-Golgi compartments such as the ER. We could not detect endogenous polypeptide GalNAc-transferase activity in the ER of HeLa cells, neither by subcellular fractionation nor by situ glycosylation of an ER-retained form of CD8 (CD8/E19). However, upon relocation of chimeric GalNAc-T1 or -T2 to the ER, CD8/E19 is glycosylated with different efficiencies indicating that all components required for initiation of O-glycosylation are present in the ER except for polypeptide GalNAc-transferases.
Röttger
S
S
Cell Biology Programme, EMBL, Heidelberg, Germany.
White
J
J
Wandall
H H
HH
Olivo
J C
JC
Stark
A
A
Bennett
E P
EP
Whitehouse
C
C
Berger
E G
EG
Clausen
H
H
Nilsson
T
T
D001483:Base Sequence; D002451:Cell Compartmentation / Q000502:physiology; D003001:Cloning, Molecular; D004721:Endoplasmic Reticulum / Q000201:enzymology; D000939:Epitopes / Q000235:genetics; D005455:Fluorescent Antibody Technique; D006031:Glycosylation; D006056:Golgi Apparatus / Q000201:enzymology* / Q000648:ultrastructure; D006367:HeLa Cells; D006801:Humans; D007527:Isoenzymes / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D017350:N-Acetylgalactosaminyltransferases / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
D000939:Epitopes; D007527:Isoenzymes; D017350:N-Acetylgalactosaminyltransferases