Abstract
The human selenoprotein thioredoxin reductase 1 (TrxR1), encoded by the TXNRD1 gene, is a key player in redox regulation. Alternative splicing generates several TrxR1 variants, one of which is v3 that carries an atypical N-terminal glutaredoxin domain. When overexpressed, v3 associates with membranes and triggers formation of filopodia. Here we found that membrane targeting of v3 is mediated by myristoylation and palmitoylation of its N-terminal MGC motif, through which v3 specifically targets membrane rafts. This was suggested by its localization in cholera toxin subunit B-stained membrane areas and also shown using lipid fractionation experiments. Utilizing site-directed mutant variants, we also found that v3-mediated generation of filopodia is independent of the Cys residues in its redox active site, but dependent upon its membrane raft targeting. These results identify v3 as an intricately regulated protein that expands TXNRD1-derived protein functions to the membrane raft compartment.
Highlights
The TXNRD1_v3 (“v3”) protein is a rare variant of human thioredoxin reductase 1
This revealed that a variant with the two Cys moieties of the redox active site changed to redox inactive Ser residues (C76S/C79S) yielded an identical phenotype of membrane association as seen with wild type v3(Grx), which was highly reminiscent of that reported earlier for the wild type protein [23, 28]
These results strongly suggested that overexpressed v3 in transfected cells becomes targeted to cell membranes through myristoylation and palmitoylation, where it closely associates with the membrane raft marker cholera toxin subunit-B (CT-B)
Summary
The TXNRD1_v3 (“v3”) protein is a rare variant of human thioredoxin reductase 1. Utilizing site-directed mutant variants, we found that v3-mediated generation of filopodia is independent of the Cys residues in its redox active site, but dependent upon its membrane raft targeting These results identify v3 as an intricately regulated protein that expands TXNRD1-derived protein functions to the membrane raft compartment. It gives rise to numerous transcripts that can undergo extensive splicing, in particular at the 5Ј-end, producing several different protein isoforms (8, 9, 20 –22) One of these isoforms, TXNRD1_v3 (“v3”), is peculiar by utilizing three additional exons encoding an atypical dithiol active site Grx domain, which is expressed in N-terminal fusion to the classical TrxR1 module [8, 20, 23, 24]. These membrane structures have commonly been called “lipid rafts,” but are in the present study named and defined according to the 2006 consensus of the Keystone Symposium on Lipid Rafts and Cell Function [29]
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