Identification of a small cytoplasmic ankyrin (AnkG119) 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, AnkG119 (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. AnkG119 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 II with a highly conserved spectrin-binding motif, and a truncated 5-kD putative regulatory domain. An AnkG119 cDNA probe hybridized to a 6.0-kb message in human and rat kidney, placenta, and skeletal muscle. An antibody raised to AnkG119 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. AnkG119 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. AnkG119 immunocreativity 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 AnkG119 interacts with beta I sigma 1 spectrin in vitro with a Kd = 4.2 +/- 4.0 ( +/- 2 SD) nM, and avidly binds the beta spectrin in MDCK cell lysates. Collectively, these data identify AnkG119 as a novel small ankyrin that binds and colocalizes with beta I sigma spectrin in the ER and Golgi apparatus, and possible on a subset of endosomes during the early stages of polarity development. We hypothesize that AnkG119 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.
Devarajan
P
P
Department of Pathology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
Stabach
P R
PR
Mann
A S
AS
Ardito
T
T
Kashgarian
M
M
Morrow
J S
JS
D000595:Amino Acid Sequence; D000818:Animals; D017487:Ankyrins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002460:Cell Line; D003593:Cytoplasm / Q000378:metabolism; D017931:DNA Primers; D004285:Dogs; D006056:Golgi Apparatus / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000378:metabolism*; D018374:LLC-PK1 Cells; D051379:Mice; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D010920:Placenta / Q000378:metabolism; D011485:Protein Binding; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D013049:Spectrin / Q000378:metabolism*; D013552:Swine
C490613:ANK3 protein, human; C094681:Ank3 protein, mouse; C490614:Ank3 protein, rat; D017487:Ankyrins; D017931:DNA Primers; D010446:Peptide Fragments; D013049:Spectrin