Isoform specific regulation of divalent metal (ion) transporter (DMT1) by proteasomal degradation

Abstract

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.

Figure 1

Parkin does not target 1A/+IRE DMT1. Keys for the levels of doxycycline used to induce DMT1 expression (0 or 25 nM) and the Parkin construct used for transient transfection (wt, T240R or none = empty vector) are present between parts A & C and parts B & D. A. Blot probed with anti-DMT1 directed against the 4^th extracellular region. B. Blot probed with anti-DMT1 directed against exons 2–3. In parts A and B, the lowest DMT1 band is ~75kDa. C. Blot from A after it was stripped and reprobed with anti-actin (load control). The actin band is ~45 kDa. There was some ineffective transfer in the high molecular weight region of the left lane. D. Blot from B after it was stripped and reprobed with anti-FLAG (tests for successful transient transfection with FLAG-tagged-Parkin construct). The FLAG-tagged-Parkin band is ~50 kDa; it is absent from the 3^rd and 6^th lanes because the DNA construct was empty vector.

Figure 2

Parkin does target 1B/−IRE DMT1. Keys for the levels of doxycycline used to induce DMT1 expression (0 or 25 nM) and the Parkin construct used for transient transfection (wt, T240R or none = empty vector) are present between parts A & C and parts B & D. A. Blot probed with anti-DMT1 directed against the 4^th extracellular region. B. Blot probed with anti-DMT1 directed against exons 2–3. In parts A and B, the lowest DMT1 band is ~75kDa. C. Blot from A after it was stripped and reprobed with anti-actin (load control). The actin band is ~45 kDa. D. Blot from B after it was stripped and reprobed with anti-FLAG (tests for successful transient transfection with FLAG-tagged-Parkin construct). The FLAG-tagged-Parkin band is ~50 kDa; it is absent from the 3^rd and 6^th lanes because the DNA construct was empty vector.

Figure 3

How E3-ligases could target DMT1 isoforms. All 4 parts depict the 4 DMT1 isoforms in a tabular array: 1A isoforms above 1B; +IRE isoforms to the left of −IRE. Large lines strike through the isoforms that are / might be degraded. A. Parkin targets 1B DMT1. B. Ndfips might target 1A DMT1. C. Ndfips might target +IRE DMT1. D. Ndfips might target all 4 DMT1 isoforms.