Two distinct domains of protein 4.1 critical for assembly of functional nuclei in vitro.
Protein 4.1R, a multifunctional structural protein, acts as an adaptor in mature red cell membrane skeletons linking spectrin-actin complexes to plasma membrane-associated proteins. In nucleated cells protein 4.1 is not associated exclusively with plasma membrane but is also detected at several important subcellular locations crucial for cell division. To identify 4.1 domains having critical functions in nuclear assembly, 4.1 domain peptides were added to Xenopus egg extract nuclear reconstitution reactions. Morphologically disorganized, replication deficient nuclei assembled when spectrin-actin-binding domain or NuMA-binding C-terminal domain peptides were present. However, control variant spectrin-actin-binding domain peptides incapable of binding actin or mutant C-terminal domain peptides with reduced NuMA binding had no deleterious effects on nuclear reconstitution. To test whether 4.1 is required for proper nuclear assembly, 4.1 isoforms were depleted with spectrin-actin binding or C-terminal domain-specific antibodies. Nuclei assembled in the depleted extracts were deranged. However, nuclear assembly could be rescued by the addition of recombinant 4.1R. Our data establish that protein 4.1 is essential for nuclear assembly and identify two distinct 4.1 domains, initially characterized in cytoskeletal interactions, that have crucial and versatile functions in nuclear assembly.
Krauss
Sharon Wald
SW
Department of Subcellular Structure, Life Sciences Division, University of California, Lawrence Berkeley National Laboratory, 94720, USA. sakrauss@lbl.gov
Heald
Rebecca
R
Lee
Gloria
G
Nunomura
Wataru
W
Gimm
J Aura
JA
Mohandas
Narla
N
Chasis
Joel Anne
JA
D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001973:Bromodeoxyuridine / Q000494:pharmacology; D002467:Cell Nucleus / Q000378:metabolism; D003598:Cytoskeletal Proteins; D005091:Exons; D019084:Fluorescent Antibody Technique, Indirect; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation; D009479:Neuropeptides; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D014981:Xenopus
D000199:Actins; D003598:Cytoskeletal Proteins; D008565:Membrane Proteins; D009479:Neuropeptides; C028841:erythrocyte membrane band 4.1 protein; C120557:erythrocyte membrane protein band 4.1-like 1; D001973:Bromodeoxyuridine