Isoform specific regulation of divalent metal (ion) transporter (DMT1) by proteasomal degradation.
Divalent metal ion transporter (DMT1) is the major transporter for iron entrance into mammalian cells and iron exit from endosomes during the transferrin cycle. Four major mRNA isoforms correspond to four protein isoforms, differing at 5'/3' and N-/C-termini, respectively. Isoforms are designated 1A versus 1B reflecting where transcription starts or +iron responsive element (+IRE) versus -IRE reflecting the presence/absence of an IRE in the 3' end of the mRNA. These differences imply regulation at transcriptional and posttranscriptional levels. Many proteins are degraded by a ubiquitination-dependent mechanism. Two different ubiquitin ligases (E3s) appear to be involved in DMT1 ubiquitination: Parkin or neuronal precursor cell-expressed developmentally downregulated 4 (Nedd4) family E3s which often utilize Nedd4 family interacting protein-1 and -2 (Ndfip1 and 2) to ubiquitinate their substrate proteins. Prior data suggest that Parkin ubiquitinates 1B DMT1 but not 1A DMT1 while Nedd4/Ndfips ligate ubiquitin to DMT1 in the duodenum where 1A/+IRE DMT1 predominates. Our assay for whether these systems target DMT1 depends on two HEK293 cell lines that express permanently transfected 1A/+IRE DMT1 or 1B/-IRE DMT1 after induction by doxycycline. Transient transfection with a Parkin construct before induction diminishes 1B/-IRE DMT1 detected by immune-blots but not 1A/+IRE DMT1. Mutant Parkin serves as a control that does not affect DMT1 levels. Thus DMT1 regulation in an isoform specific fashion can occur by ubiquitination and the events involved have implications for DMT1 function and disease processes.
Department of Biochemistry, University at Buffalo, Buffalo, NY, USA. mgarrick@buffalo.edu
Michael D
MD
Garrick
Lin
L
Zhao
Jerome A
JA
Roth
Houbo
H
Jiang
Jian
J
Feng
Natalie J
NJ
Foot
Hazel
H
Dalton
Sharad
S
Kumar
Laura M
LM
Garrick
D002352:Carrier Proteins / Q000378:metabolism; D027682:Cation Transport Proteins / Q000378:metabolism*; D002460:Cell Line; D056827:Endosomal Sorting Complexes Required for Transport / Q000378:metabolism; D006801:Humans; D007501:Iron / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D000075702:Nedd4 Ubiquitin Protein Ligases; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D020033:Protein Isoforms / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism; D000099236:Solute Carrier Family 11, Member 2
D002352:Carrier Proteins; D027682:Cation Transport Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D007501:Iron; D008565:Membrane Proteins; D000075702:Nedd4 Ubiquitin Protein Ligases; D046988:Proteasome Endopeptidase Complex; D020033:Protein Isoforms; D025801:Ubiquitin; D044767:Ubiquitin-Protein Ligases; D000099236:Solute Carrier Family 11, Member 2; C451223:NDFIP1 protein, human; C477747:NDFIP2 protein, human; C000618483:Nedd4 protein, human