IDENTIFICATION OF A SMALL CYTOPLASMIC ANKYRIN (ANK(G119)) IN THE KIDNEY AND MUSCLE THAT BINDS BETA-I-SIGMA SPECTRIN AND ASSOCIATES WITH THEGOLGI-APPARATUS

Ankyrins are a family of large, membrane-associated proteins that medi ate the linkage of the cytoskeleton to a variety of membrane transport and receptor proteins. A repetitive 33-residue motif characteristic o f domain I of ankyrin has also been identified in proteins involved wi th cell cycle control and development. We have cloned and characterize d a novel ankyrin isoform, Ank(G119) (GenBank accession No. U43965), f rom the human kidney which lacks part of this repetitive domain and as sociates in MDCK cells with beta I Sigma spectrin and the Golgi appar atus, but not the plasma membrane. Sequence comparison reveals this an kyrin to be an alternative transcript of AnkG, a much larger ankyrin r ecently cloned from brain. AnkG(119) has a predicted size of 119,201 D , and contains a 47-kD domain I consisting of 13 ankyrin repeat units, a 67-kD domain IT with a highly conserved spectrin-binding motif, and a truncated 5-kD putative regulatory domain. An Ank(G119) cDNA probe hybridized to a 6.0-kb message in human and rat kidney, placenta, and skeletal muscle. An antibody raised to Ank(G119) recognized an apparen t 116-kD peptide in rat kidney cortical tissue and MDCK cell lysates, and did not react with larger isoforms of ankyrin at 190 and 210 kD in these tissues, nor in bovine brain, nor with ankyrin from human eryth rocytes. Ank(G119) remains extractable in 0.5% Triton X-100, and assum es a punctuate cytoplasmic distribution in mature MDCK cells, in contr ast to the Triton-stable plasma membrane localization of all previousl y described renal ankyrins. Ank(G119) immunoreactivity in subconfluent MDCK cells distributes with the Golgi complex in a pattern coincident with beta-COP and beta I Sigma spectrin immunoreactivity. A fusion p eptide containing residues 669-860 of Ank(G119) interacts with beta I Sigma spectrin in vitro with a K-d = 4.2 +/- 4.0 (+/-2 SD) nM, and av idly binds the beta spectrin in MDCK cell lysates. Collectively, these data identify AnkG,,, as a novel small ankyrin that binds and colocal izes with beta I Sigma spectrin in the ER and Golgi apparatus, and po ssibly on a subset of endosomes during the early stages of polarity de velopment. We hypothesize that Ank(G119) and beta I spectrin form a ve sicular Golgi-associated membrane skeleton, promote the organization o f protein microdomains within the Golgi and trans-Golgi networks, and contribute to polarized vesicle transport.