Identification of a Small Cytoplasmic Ankyrin (AnkG119) in the Kidney and Muscle That Binds βIΣ Spectrin and Associates with the Golgi Apparatus

• Published in: The Journal of cell biology 1996-05, Vol.133 (4), p.819-830
• Main Authors: Devarajan, Prasad, Stabach, Paul R., Mann, Andrea S., Ardito, Thomas, Kashgarian, Michael, Morrow, Jon S.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Ankyrins; Ankyrins - genetics; Ankyrins - metabolism; Antibodies; Base Sequence; Cell Line; Cell membranes; Cells; Cellular biology; Cytoplasm - metabolism; DNA Primers; Dogs; Epithelial cells; Erythrocytes; Golgi Apparatus - metabolism; Humans; Kidney - metabolism; Kidney cells; Kidneys; LLC PK1 cells; Membranes; Mice; Molecular Sequence Data; Muscle, Skeletal - metabolism; Peptide Fragments - metabolism; Placenta - metabolism; Protein Binding; Protein isoforms; Proteins; Rabbits; Rats; Rats, Sprague-Dawley; Spectrin - metabolism; Swine
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.133.4.819

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, AnkG119(GenBank accession No. U43965), from the human kidney which lacks part of this repetitive domain and associates in MDCK cells with βIΣ* 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. AnkG119has a predicted size of 119,201 D, and contains a 47-kD domain I consisting of 13 ankyrin repeat units, a 67-kD domain II with a highly conserved spectrin-binding motif, and a truncated 5-kD putative regulatory domain. An AnkG119cDNA probe hybridized to a 6.0-kb message in human and rat kidney, placenta, and skeletal muscle. An antibody raised to AnkG119recognized 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. AnkG119remains extractable in 0.5% Triton X-100, and assumes a punctuate cytoplasmic distribution in mature MDCK cells, in contrast to the Tritonstable plasma membrane localization of all previously described renal ankyrins. AnkG119immunoreactivity in subconfluent MDCK cells distributes with the Golgi complex in a pattern coincident with β-COP and βIΣ* spectrin immunoreactivity. A fusion peptide containing residues 669-860 of AnkG119interacts with βIΣ1 spectrin in vitro with a Kd= 4.2 ± 4.0 (±2 SD) nM, and avidly binds the β spectrin in MDCK cell lysates. Collectively, these data identify AnkG119as a novel small ankyrin that binds and colocalizes with βIΣ* spectrin in the ER and Golgi apparatus, and possibly on a subset of endosomes during the early stages of polarity development. We hypothesize that AnkG119and βI 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.