Computer-identified nuclear localization signal in exon 1A of the transporter DMT1 is essentially ineffective in nuclear targeting.
Divalent metal transporter 1 (DMT1; also called DCT1, Nramp2, or SLC11A2) has multiple isoforms that localize differently in many cell types. DMT1 +IRE species (encoded by mRNA with an iron-responsive element) are limited to the plasma membrane and cytosolic vesicles. In neural cells, -IRE isoforms of DMT1 (encoded by mRNA lacking an IRE) localize to the nucleus, plasma membrane, and cytosolic vesicles. In considering nuclear compartmentalization of -IRE isoforms, we hypothesized that the newly identified exon 1A in the N-terminus of this transporter might contain a nuclear localization signal. DNA constructs starting with exon 1A and ending with exons encoding alternative isoforms were made and transiently transfected into HEK293T and PC12 cells as well as rat sympathetic neurons. None of the constructs appeared in the nucleus despite the presence of exon 1A. Antibody specific for exon 1A was also used in both immunostaining and Western blots to investigate localization of exon 1A expressed both endogenously and ectopically in cells. Again, nuclear localization of DMT1 containing exon 1A was not observed. Our data suggest that exon 1A is neither sufficient nor necessary for DMT1 to appear in the nucleus.
Kuo
H C
HC
Department of Biochemistry, SUNY at Buffalo, Buffalo, New York 14214-3000, USA.
Smith
J J
JJ
Lis
A
A
Zhao
L
L
Gonsiorek
E A
EA
Zhou
X
X
Higgins
D M
DM
Roth
J A
JA
Garrick
M D
MD
Garrick
L M
LM
D000818:Animals; D000831:Animals, Newborn; D015153:Blotting, Western / Q000379:methods; D027682:Cation Transport Proteins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism*; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism; D004622:Embryo, Mammalian; D005091:Exons; D049452:Green Fluorescent Proteins; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D033862:Iron-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D007668:Kidney; D008164:Luminescent Proteins / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D019913:Nuclear Localization Signals / Q000378:metabolism*; D020033:Protein Isoforms / Q000378:metabolism; D021381:Protein Transport; D011506:Proteins / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D013564:Sympathetic Nervous System / Q000166:cytology*; D014162:Transfection / Q000379:methods; D000099236:Solute Carrier Family 11, Member 2
D027682:Cation Transport Proteins; D049452:Green Fluorescent Proteins; D033862:Iron-Binding Proteins; D008164:Luminescent Proteins; D019913:Nuclear Localization Signals; D020033:Protein Isoforms; D011506:Proteins; D012333:RNA, Messenger; D000099236:Solute Carrier Family 11, Member 2