Isolation and characterization of the Golgi apparatus of a flagellate scaly green alga.
Highly purified Golgi membranes were isolated from the scaly green flagellate Scherffelia dubia using osmotic shock for controlled cell rupture, differential centrifugations and a discontinuous sucrose density gradient centrifugation. Three Golgi membrane fractions (based on the distribution of IDPase activity in the gradient) at densities 1.14 g/ml, 1.17 g/ml and 1.20 g/ml were obtained. The specific IDPase activity in these fractions was enriched about 78-fold compared to the crude cell homogenate. The Golgi membrane fractions were further characterized by electron microscopy, SDS-PAGE and lectin blotting. The low density fraction (1.14 g/ml) contained two distinct vesicle populations and scale precursors associated with the outer surface of the larger-size vesicles. The medium density fraction (1.17 g/ml) contained in addition to the larger vesicles, multilamellate vesicles and semicircular cisternae. Finally, in the high density fraction (1.20 g/ml) in addition to small and large vesicles, a tubular membrane reticulum was observed. The three Golgi membrane fractions revealed the same complex overall polypeptide composition when analyzed by SDS-PAGE, but gradual quantitative differences in the polypeptide profile between fractions were observed. The lectins GNA, DSA, and AAA bound to several glycoproteins in all Golgi membrane fractions. Deglycosylation with N-glycosidase F showed that all carbohydrate structures recognized by GNA and DSA, and one recognized by AAA were of the N-glycosidic type indicating the presence of both "high mannose" and "processed" N-glycans in the Golgi apparatus of S. dubia.
Grunow
A
A
Botanisches Institut, Lehrstuhl I, Universität zu Köln, Germany.
Becker
B
B
Melkonian
M
M
D002499:Centrifugation, Density Gradient; D000460:Chlorophyta / Q000648:ultrastructure*; D004591:Electrophoresis, Polyacrylamide Gel; D005407:Flagella; D006056:Golgi Apparatus / Q000648:ultrastructure*; D007425:Intracellular Membranes / Q000648:ultrastructure*; D037102:Lectins / Q000378:metabolism; D008854:Microscopy, Electron; D008861:Microsomes / Q000648:ultrastructure; D009997:Osmotic Pressure; D013347:Subcellular Fractions / Q000737:chemistry
D037102:Lectins