Two forms of the apoptosis-linked protein ALG-2 with different Ca(2+) affinities and target recognition.
The apoptosis-linked gene ALG-2 encodes a Ca(2+)-binding protein of the penta EF-hand family. To investigate the Ca(2+) binding properties of the recombinant ALG-2 protein, we have cloned ALG-2 cDNA from mouse liver mRNA. Sequence analysis showed that two types of clones were present. One (named ALG-2,5) corresponds to the published ALG-2 sequence (Vito, P., Lacana, E., and D'Adamio, L. (1996) Science 271, 521-525); the second (named ALG-2,1) is 6 nucleotides shorter, and the corresponding protein lacks the amino acid residues Gly(121) and Phe(122). Both transcripts are present in mouse tissues in the same 2:1 molar ratio. The ALG-2,5 and ALG-2,1 recombinant proteins are fully soluble in the metal-free form but can be precipitated from bacterial lysates by Ca(2+). In the presence of Tween the Ca(2+) binding profiles display two high affinity sites with [Ca(2+)](0.5) values of 1.2 and 3.1 microM for ALG-2,5 and ALG-2,1, respectively, plus one low affinity site. Using the yeast two-hybrid system we demonstrate that both proteins have a strong tendency to form homo- and heterodimers. In contrast to ALG-2, 5, the ALG-2,1 isoform does not interact with the target protein AIP-1, earlier described to play a role in apoptosis (Vito, P., Pellegrini, L., Guiet, C., and D'Adamio, L. (1999) J. Biol. Chem. 274, 1533-1540). We propose that the minor sequence difference between ALG-2,5 and ALG-2,1 affects the Ca(2+) binding properties and function of the proteins.
Tarabykina
S
S
Department of Molecular Cell Biology, Institute of Molecular Biology, University of Copenhagen, Oster Farimagsgade 2A, Copenhagen, DK-1353, Denmark.
Møller
A L
AL
Durussel
I
I
Cox
J
J
Berchtold
M W
MW
D000818:Animals; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins; D001921:Brain / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D005260:Female; D014644:Genetic Variation; D007668:Kidney / Q000378:metabolism; D008099:Liver / Q000378:metabolism*; D051379:Mice; D010053:Ovary / Q000378:metabolism; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D014158:Transcription, Genetic
D051017:Apoptosis Regulatory Proteins; D002135:Calcium-Binding Proteins; C490815:Pdcd6 protein, mouse; D020033:Protein Isoforms; D012333:RNA, Messenger; D011994:Recombinant Proteins; D002118:Calcium