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 skelet on protein spectrin, we have identified an isoform of ankyrin that ass ociates at steady state with the Golgi complex. Immune-light and -elec tron microscopy show that this ankyrin isoform localizes to the perinu clear cytoplasm on tubular vesicular structures that co-stain with Gol gi marker proteins. An antiserum raised against erythrocyte ankyrin, w hich was used to identify the Golgi ankyrin, recognized three prominen t polypeptides of 220, 213 and 195 kDa in MDCK cells. Affinity purific ation of this antiserum against each of these MDCK cell ankyrins revea led that only an antibody specific for the 195 kDa form retained the a bility to stain the Golgi complex; affinity purified antibody preparat ions specific for both the 220 and 213 kDa forms stained punctate and reticular cytoplasmic structures distinct from the Golgi complex. Anti body specific for the 195 kDa ankyrin did not recognize a recently ide ntified 119 kDa ankyrin that is also localized to the Golgi. The 195 k Da Golgi ankyrin binds purified erythrocyte spectrin, and rapidly co-s ediments with Golgi beta-spectrin during brief, low speed centrifugati on of Triton X-100 extracts of MDCK cells. Golgi ankyrin and beta-spec trin are retained on tubular vesicular 'Golgi ghosts' following extrac tion of cultured cells,vith Triton X-100. Significantly, Golgi ghost t ubules containing ankyrin/spectrin are colinear with individual microt ubules, suggesting a role for both Golgi membrane skeleton and microtu bules in spatial localization of the Golgi. Golgi ankyrin dissociates from Golgi membranes during mitosis and in cells treated with brefeldi n A, indicating that Golgi ankyrin has a dynamic assembly state simila r to that of Golgi spectrin and other Golgi membrane coat proteins.