We have reported here the use of two independent methods to demonstrate the association of B3GNT1 and B4GALT1 within the secretory pathway. Both coimmunoprecipitation and cellular relocalization assays provide evidence for an interaction between these two enzymes. The physical association between B3GNT1 and B4GALT1 likely contributes to the functional coupling of these two enzymes in producing the polyLacNAc glycan structure.
The distinctive structures of glycosyltransferases, namely their common type II transmembrane orientation, made it possible to adapt the ER retention assay (Nilsson et al. 1994
) by employing a C-terminal KDEL tag for recruitment. C-terminal KDEL tags are well-established cellular signals for soluble protein retention to the ER (Pelham 1990
) and have been exploited previously to relocalize type II transmembrane proteins to the ER (Munro 1995
). Our data show that the KDEL tag can be applied to other Golgi glycosyltransferases and used to retain trans
-Golgi glycosyltransferases and their partners in the ER.
Recent reports have suggested that Golgi localization of glycosyltransferases can depend on native cytoplasmic signals (Grabenhorst and Conradt 1999
; Milland et al. 2001
; Giraudo and Maccioni 2003b
; Schmitz et al. 2008
; Tu et al. 2008
). While the original p33-mediated ER retention assay was able to detect interactions between medial glycosyltransferases (Nilsson et al. 1994
), the use of a KDEL motif has advantages over the original system. First, the KDEL fusion requires a smaller overall change to the chimeric protein: fusion of 6 amino acids (SEKDEL), compared to the addition of the 47 amino acids that comprise the cytoplasmic domain of the p33 protein. Second, the addition of a KDEL motif does not involve replacement of any amino acids. Lastly, the KDEL motif does not necessitate the removal of glycosyltransferase cytoplasmic domains that may contribute to protein localization. The efficiency of partner protein ER-relocalization is likely to depend on the individual glycosyltransferases studied, and the p33 system may be more effective than the KDEL motif for certain enzyme pairs.
It is worth noting that the relative efficiency of relocalization for trans
-Golgi partners versus medial
Golgi partners cannot be compared directly since the different native pH environments may influence the ability of such partners to achieve ER-relocalization. Indeed, Colley and co-workers demonstrated that β-galactoside α-2,6-sialyltransferase 1, a trans
-Golgi resident enzyme, formed insoluble oligomers only when harvested under pH conditions similar to those found at the trans
-Golgi (Chen et al. 2000
). The same pH gradient may explain why the KDEL-mediated ER retention assay reported here did not result in the relocalization of 100% of the examined trans
Probing lysates of cells transfected with B4GALT1-HA using an anti-HA antibody revealed two isoforms: one at the expected molecular weight for unmodified B4GALT1-HA (45 kDa) and another at a higher molecular weight (approximately 53 kDa) (Figure B, lanes 2, 5, 7). We hypothesize that this higher molecular weight band represents the presence of an extended glycan modification added during passage through the Golgi. This hypothesis is consistent with the observation that lysates from cells transfected with B4GALT1-HA-KDEL contain predominantly the lower molecular weight form (Figure B, lanes 3, 6, 8), since the KDEL-tagged isoform is less likely to traffic through the Golgi and be modified by glycan extending enzymes. We were intrigued to observe that B3GNT1-myc and B3GNT1-myc-KDEL both preferentially precipitated the higher molecular weight isoform of B4GALT1-HA-KDEL (Figure B, lanes 14 and 16). This finding suggests that an extended glycan on B4GALT1 may enhance binding to B3GNT1.
B4GALT1 transmembrane residues Cys29 and His32 are required for Golgi retention and appear to also contribute to B4GALT1 homodimerization (Aoki et al. 1992
; Yamaguchi and Fukuda 1995
). An intriguing possibility is that these same residues might also mediate association between B4GALT1 and B3GNT1. Also noteworthy is B3GNT1's unusually long, 28-amino-acid transmembrane domain, which is particularly unexpected in a trans
-Golgi-resident glycosyltransferase. Perhaps an interaction between B3GNT1 and B4GALT1 allows B3GNT1 to take advantage of the more canonical B4GALT1 transmembrane domain to stabilize B3GNT1 localization to the trans
-Golgi. Future experiments will be needed to test these possibilities directly. Furthermore, experiments using purified components will help to address whether the interaction observed is direct.
The present study has focused on two of the main enzymes that cooperate in polyLacNAc synthesis. However, other galactosyltransferases also participate in this biosynthetic process (Lo et al. 1998
; Hennet 2002
). Preliminary results using the ER recruitment assay described here provide evidence for an association between B3GNT1 and UDP-Gal:βGlcNAc β-1,4-galactosyltransferase, polypeptide 4, B4GALT4, in cotransfected cells (data not shown). In vitro, B4GALT1 has been shown to display substrate preference for N
-linked and core 4 O
-linked glycans while B4GALT4 has substrate preference for core 2 O
-linked glycans (Ujita et al. 2000
). B4GALT1 and B4GALT4 might therefore compete for association with B3GNT1 and bias B3GNT1 substrate preference in vivo. More detailed information about the molecular interfaces of B3GNT1: B4GALT complexes will aid in investigating this hypothesis. Alternatively, B3GNT1, B4GALT1, and B4GALT4 may associate with each other within a single, large heterocomplex that allows B3GNT1 to process both N
-linked and O
-linked glycans. By disrupting or strengthening associations between B3GNT1 and B4GALT1 or between B3GNT1 and B4GALT4, it may be possible to alter the glycan synthetic capacity in cells. Furthermore, engineering an enzyme containing both B3GNT1 and B4GALT1 catalytic domains may generate increased polyLacNAc length by increasing the processivity of the reaction and allow further studies on how polyLacNAc length affects its biological activities.
In conclusion, we have shown the first example of enzyme association among trans-Golgi-localized glycosyltransferases using an ER retention assay that does not alter the N-terminal cytoplasmic, transmembrane, and stem domains of these type II transmembrane proteins. The physical association between these two glycosyltransferases likely contributes to the localization of both B3GNT1 and B4GALT1 to the trans-Golgi and, importantly, to regulating the production of polyLacNAc from these enzymes.