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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.

An affinity-purified, monospecific rabbit antibody against soluble human milk galactosyltransferase was used to localize the enzyme in HeLa cells by immunofluorescence and by the protein A-gold technique at the electron microscope level. Specific immunofluorescence was observed in a juxtanuclear cytoplasmic region which was identified, on immunostained thin sections of lowtemperature Lowicryl K4M-embedded HeLa cells, as Golgi apparatus. Label by gold particles was limited to two to three trans cisternae of the Golgi apparatus, indicating a compartmentalization of galactosyltransferase in the cisternal stack. Combination of preembedding thiamine pyrophosphatase cytochemistry with postembedding immunostaining for galactosyltransferase proved codistribution of the two enzymes. However, the acid phosphatase-positive, trans-most cisterna was negative for galactosyltransferase . The close topological association of both galactosyltransferase and thiamine pyrophosphatase (or nucleoside diphosphatase) suggests a concerted action of both enzymes in glycosylation.Biosynthesis of complex type heteroglycans proceeds in two distinct stages in different cellular organelles: first, N-glycosylation involving the en bloc transfer of a lipid-linked oligosaccharide takes place in the rough endoplasmic reticulum (for recent review, see reference 27) where part of it is processed by glucosidases (6); thereafter, the glycoproteins move to the Golgi apparatus where N-linked glycans are further processed (10) before being elongated by sequential action of terminal glycosyltransferases which include N-acetyl-D-glucosaminyl-, galactosyl-, fucosyl-, and sialyltransferases (for review, see reference 26). On the basis of cell fractionation studies (3,5, 11,15) and autoradiographic (1, 19) evidence, chain elongation is assumed to occur in the Golgi apparatus. Accordingly, galactosyltransferase activity has frequently been selected as marker for Golgi fractions (see 3,5,8, 11,14,15,26 for selected references). In order to obtain conclusive in situ evidence of the Golgi-association of galactosyltransferase (UDP-galactose: ,ß-D-N-acetylglucosaminyl-protein 8 (1-4) transferase, EC 2.4 . (23,24). We report here the first ultrastructural localization of a glycosyltransferase, namely galactosyltransferase, and demonstrate its presence in a distinct Golgi subcompartment which is composed of two to three thiamine pyrophosphatase-positive transGolgi cisternae. MATERIALS AND METHODSGalactosyltransferase was purified from pooled human milk (9). The immunization schedule included six subcutaneous injections of 0.2 mg of galactosyltransferase emulsified in complete Freund's adjuvant into rabbits. Antibody production was monitored by an enzyme-linked immunosorbent assay (ELISA) (2). Monospecific antibodies were obtained by affinity purification on immobilized galactosyltransferase. Their specificity towards purified galactosyltransferase was checked by ELISA (2) and the immunoreplica technique essentially according to Towbin et al. (29), except th...

Abstract. Immunoelectron microscopy and stereology were used to identify and quantitate Golgi fragments in metaphase HeLa cells and to study Golgi reassembly during telophase. On ultrathin frozen sections of metaphase cells, labeling for the Golgi marker protein, galactosyltransferase, was found over multivesicular Golgi clusters and free vesicles that were found mainly in the mitotic spindle region. The density of Golgi cluster membrane varied from cell to cell and was inversely related to the density of free vesicles in the spindle. There were thousands of free Golgi vesicles and they comprised a significant proportion of the total Golgi membrane.During telophase, the distribution of galactosyltransferase labeling shifted from free Golgi vesicles towards Golgi clusters and the population of free vesicles was depleted. The number of clusters was no more than in metaphase cells so the observed fourfold increase in membrane surface meant that individual clusters had increased in size. More than half of these had cisterna(e) and were located next to "buds" on the endoplasmic reticulum. Early in G1 the number of clusters dropped as they congregated in the juxtanuclear region and fused.These results show that fragmentation of the Golgi apparatus yields Golgi clusters and free vesicles and reassembly from these fragments is at least a two-step process: (a) growth of a limited number of dispersed clusters by accretion and fusion of vesicles to form cisternal clusters next to membranous "buds" on the endoplasmic reticulum; (b) congregation and fusion to form the interphase Golgi stack in the juxtanuclear region.