Golgi membrane skeleton: identification, localization and oligomerization of a 195 kDa ankyrin isoform associated with the Golgi complex.
To extend our finding of a Golgi-localized form of the membrane skeleton protein spectrin, we have identified an isoform of ankyrin that associates at steady state with the Golgi complex. Immuno-light and -electron microscopy show that this ankyrin isoform localizes to the perinuclear cytoplasm on tubular vesicular structures that co-stain with Golgi marker proteins. An antiserum raised against erythrocyte ankyrin, which was used to identify the Golgi ankyrin, recognized three prominent polypeptides of 220, 213 and 195 kDa in MDCK cells. Affinity purification of this antiserum against each of these MDCK cell ankyrins revealed that only an antibody specific for the 195 kDa form retained the ability to stain the Golgi complex; affinity purified antibody preparations specific for both the 220 and 213 kDa forms stained punctate and reticular cytoplasmic structures distinct from the Golgi complex. Antibody specific for the 195 kDa ankyrin did not recognize a recently identified 119 kDa ankyrin that is also localized to the Golgi. The 195 kDa Golgi ankyrin binds purified erythrocyte spectrin, and rapidly co-sediments with Golgi beta-spectrin during brief, low speed centrifugation of Triton X-100 extracts of MDCK cells. Golgi ankyrin and beta-spectrin are retained on tubular vesicular 'Golgi ghosts' following extraction of cultured cells with Triton X-100. Significantly, Golgi ghost tubules containing ankyrin/spectrin are co-linear with individual microtubules, suggesting a role for both Golgi membrane skeleton and microtubules in spatial localization of the Golgi. Golgi ankyrin dissociates from Golgi membranes during mitosis and in cells treated with brefeldin A, indicating that Golgi ankyrin has a dynamic assembly state similar to that of Golgi spectrin and other Golgi membrane coat proteins.
Beck
K A
KA
Department of Molecular and Cellular Physiology, Stanford University School of Medicine, CA 94305-5426, USA.
Buchanan
J A
JA
Nelson
W J
WJ
D000818:Animals; D017487:Ankyrins / Q000737:chemistry / Q000378:metabolism*; D000918:Antibody Specificity; D020126:Brefeldin A; D002460:Cell Line; D003517:Cyclopentanes / Q000494:pharmacology; D004285:Dogs; D004912:Erythrocytes / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism* / Q000648:ultrastructure*; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism / Q000648:ultrastructure; D016253:Microscopy, Immunoelectron; D008870:Microtubules / Q000378:metabolism / Q000648:ultrastructure; D008938:Mitosis; D008970:Molecular Weight; D051381:Rats; D013049:Spectrin / Q000378:metabolism
D017487:Ankyrins; D003517:Cyclopentanes; D013049:Spectrin; D020126:Brefeldin A