Pharmacological agents have proven useful for gaining fundamental insights into the biology of the Golgi apparatus. This review summarizes pertinent and recent work on the effects on this organelle of monensin, brefeldin A, bafilomycin, ilimaquinone, okadaic acid, retinoic acid, and nocodazole. The molecular targets of monensin, brefeldin A, ilimaquinone, and retinoic acid remain to be elucidated whereas those for bafilomycin (vacuolar H+-ATPase), okadaic acid (serine/threonine phosphatases types 1, 2a, and 2b), and nocodazole (microtubules) are reasonably well understood. The molecular target of brefeldin has not been defined, but has been suggested to involve guanine nucleotide exchange proteins acting on ADP-ribosylation factor 1. Whether a defined molecular target can be found for monensin must be questioned since its main action consists in exchanging protons for Na+ which leads to osmotic swelling of post-Golgi endosomal structures and Golgi subcompartments by virtue of its membrane-associated effect as a cationophore. Brefeldin A was one of the most thoroughly investigated Golgi-disturbing agents and proved instrumental in unraveling retrograde flow mechanisms in the secretory pathways. Okadaic acid attracted interest for its properties mimicking mitotic fragmentation of the Golgi apparatus. Nocodazole was instrumental in establishing the cytoskeletal anchoring of the Golgi apparatus close to the microtubular organizing center.
Based on the detection of expressed sequence tags that are similar to known galactosyltransferase sequences, we have isolated three novel UDP-galactose:-N-acetylglucosamine 1,3-galactosyltransferase (3GalT) genes from a mouse genomic library. The three genes, named 3GalT-I, -II, and -III, encode type II transmembrane proteins of 326, 422, and 331 amino acids, respectively. The three proteins constitute a distinct subfamily as they do not share any sequence identity with other eucaryotic galactosyltransferases. Also, the entire protein-coding region of the three 3GalT genes was contained in a single exon, which contrasts with the genomic organization of the 1,4-and ␣1,3-galactosyltransferase genes. The three 3GalT genes were mainly expressed in brain tissue. The expression of the fulllength murine genes as recombinant baculoviruses in insect cells revealed that the 3GalT enzymes share the same acceptor specificity for -linked GlcNAc, although they differ in their K m for this acceptor and the donor UDP-Gal. The identification of 3GalT genes emphasizes the structural diversity present in the galactosyltransferase gene family.
Human and mouse cDNAs encoding a new β-1,3- N -acetylglucosaminyltransferase (β3GnT) have been isolated from fetal and newborn brain libraries. The human and mouse cDNAs included ORFs coding for predicted type II transmembrane polypeptides of 329 and 325 aa, respectively. The human and mouse β3GnT homologues shared 90% similarity. The β3GnT gene was widely expressed in human and mouse tissues, although differences in the transcript levels were visible, thus indicating possible tissue-specific regulation mechanisms. The β3GnT enzyme showed a marked preference for Gal(β1–4)Glc(NAc)-based acceptors, whereas no activity was detected on type 1 Gal(β1–3)GlcNAc and O-glycan core 1 Gal(β1–3)GalNAc acceptors. The new β3GnT enzyme was capable of both initiating and elongating poly- N -acetyllactosamine chains, which demonstrated its identity with the poly- N -acetyllactosamine synthase enzyme (E.C. 2.4.1.149), showed no similarity with the i antigen β3GnT enzyme described recently, and, strikingly, included several amino acid motifs in its protein that have been recently identified in β-1,3-galactosyltransferase enzymes. The comparison between the new UDP–GlcNAc:βGal β3GnT and the three UDP–Gal:βGlcNAc β-1,3-galactosyltransferases-I, -II, and -III reveals glycosyltransferases that share conserved sequence motifs though exhibiting inverted donor and acceptor specificities. This suggests that the conserved amino acid motifs likely represent residues required for the catalysis of the glycosidic (β1–3) linkage.
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