Identification of a small cytoplasmic ankyrin (Ank(G119)) in the kidney and muscle that binds beta I Sigma spectrin and associates with the Golgi apparatus

Ankyrins are a family of large, membrane-associated proteins that mediate the linkage of the cytoskeleton to a variety of membrane transport and receptor proteins. A repetitive 33-residue motif characteristic of domain I of ankyrin has also been identified in proteins involved with cell cycle control and development. We have cloned and characterized a novel ankyrin isoform, Ank(G119) (GenBank accession No. U43965), from the human kidney which lacks part of this repetitive domain and associates in MDCK cells with beta I Sigma* spectrin and the Golgi apparatus, but not the plasma membrane. Sequence comparison reveals this ankyrin to be an alternative transcript of AnkG, a much larger ankyrin recently 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 apparent 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 erythrocytes. Ank(G119) remains extractable in 0.5% Triton X-100, and assumes a punctuate cytoplasmic distribution in mature MDCK cells, in contrast to the Triton-stable plasma membrane localization of all previously 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 peptide 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 avidly binds the beta spectrin in MDCK cell lysates. Collectively, these data identify AnkG,,, as a novel small ankyrin that binds and colocalizes with beta I Sigma* spectrin in the ER and Golgi apparatus, and possibly on a subset of endosomes during the early stages of polarity development. We hypothesize that Ank(G119) and beta I spectrin form a vesicular Golgi-associated membrane skeleton, promote the organization of protein microdomains within the Golgi and trans-Golgi networks, and contribute to polarized vesicle transport.