The N-terminal 209-aa domain of high molecular-weight 4.1R isoforms abrogates 4.1R targeting to the nucleus.
An extensive repertoire of protein 4.1R isoforms is predominantly generated by alternative pre-mRNA splicing and differential usage of two translation initiation sites. The usage of the most upstream ATG (ATG-1) generates isoforms containing N-terminal extensions of up to 209 aa compared with those translated from the downstream ATG (ATG-2). To characterize nonerythroid 4.1R proteins translated from ATG-1 and analyze their intracellular localization, we cloned 4.1R cDNAs containing this translation initiation site. Six different clones were isolated from the nucleated human MOLT-4 T-cell line by reverse transcriptase-PCR techniques. Transient expression of the six ATG-1-translated 4.1R isoforms tagged with a c-Myc epitope revealed that all of them predominantly distributed to the plasma membrane and the endoplasmic reticulum. Staining of MOLT-4 cell plasma membranes but not nuclei was also observed by immunofluorescence microscopy by using an antibody specific to the N-terminal extension. Consistent with this, the antibody reacted with a major endogenous protein of approximately 145 kDa present in nonnuclear but absent from nuclear fractions prepared from MOLT-4 cells. Because these data suggested that ATG-1-translated 4.1R isoforms were predominantly excluded from the nucleus, we fused the 209-aa domain to nuclear 4.1R isoforms encoded from ATG-2 and observed that this domain inhibited their nuclear targeting. All these results indicate that the N-terminal domain of ATG-1-translated 4.1R isoforms plays a pivotal role in differential targeting of proteins 4.1R.
Luque
C M
CM
Centro de Biología Molecular "Severo Ochoa" (Consejo Superior de Investigaciones Científicas/UAM), Universidad Autónoma de Madrid, E-28049 Madrid, Spain.
Lallena
M J
MJ
Pérez-Ferreiro
C M
CM
de Isidro
Y
Y
De Cárcer
G
G
Alonso
M A
MA
Correas
I
I
D001692:Biological Transport; D002451:Cell Compartmentation / Q000502:physiology*; D002467:Cell Nucleus / Q000378:metabolism*; D003001:Cloning, Molecular; D003598:Cytoskeletal Proteins; D018076:DNA, Complementary / Q000235:genetics; D008221:Lymphoid Tissue / Q000378:metabolism*; D008565:Membrane Proteins; D009479:Neuropeptides; D020033:Protein Isoforms / Q000378:metabolism; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013601:T-Lymphocytes / Q000378:metabolism*
D003598:Cytoskeletal Proteins; D018076:DNA, Complementary; D008565:Membrane Proteins; D009479:Neuropeptides; D020033:Protein Isoforms; D011506:Proteins; D011994:Recombinant Proteins; C028841:erythrocyte membrane band 4.1 protein; C120557:erythrocyte membrane protein band 4.1-like 1