SMIM1, carrier of the Vel blood group, is a tail-anchored transmembrane protein and readily forms homodimers in a cell-free system.

Antibodies to the Vel blood group antigen can cause adverse hemolytic reactions unless Vel-negative blood units are transfused. Since the genetic background of Vel-negativity was discovered in 2013, DNA-based typing of the 17-bp deletion causing the phenotype has facilitated identification of Vel-negative blood donors. SMIM1, the gene underlying Vel, encodes a 78-amino acid erythroid transmembrane protein of unknown function. The transmembrane orientation of SMIM1 has been debated since experimental data supported both the N- and C-termini being extracellular. Likewise, computational predictions of its orientation were divided and potential alternatives such as monotopic or dual-topology have been discussed but not investigated. We used a cell-free system to explore the topology of SMIM1 when synthesized in the endoplasmic reticulum (ER). SMIM1 was tagged with an opsin-derived N-glycosylation reporter at either the N- or C-terminus and synthesized in vitro using rabbit reticulocyte lysate supplemented with canine pancreatic microsomes as a source of ER membrane. SMIM1 topology was then determined by assessing the N-glycosylation of its N- or C-terminal tags. Complementary experiments were carried out by expressing the same SMIM1 variants in HEK293T/17 cells and establishing their membrane orientation by immunoblotting and flow cytometry. Our data consistently indicate that SMIM1 has its short C-terminus located extracellularly and that it most likely belongs to the tail-anchored class of membrane proteins with the bulk of the polypeptide located in the cytoplasm. Having established its membrane orientation in an independent model system, future work can now focus on functional aspects of SMIM1 as a potential regulator of erythropoiesis.

Dimerization of small integral membrane protein 1 promotes cell surface presentation of the Vel blood group epitope.

The Vel blood group antigen is carried on the short extracellular segment of the 78-amino-acid-long, type II transmembrane protein SMIM1 of unknown function. Here, using biochemical analysis and flow cytometry of cells expressing wild-type and mutant alleles of SMIM1, we demonstrate that dimerization of SMIM1 promotes cell surface display of the Vel epitope. We show that SMIM1 dimerization is mediated both by an extracellular Cys77-dependent, homomeric disulfide linkage and via a GxxxG helix-helix interaction motif in the transmembrane domain. These results provide important context for the observed variability in reactivity patterns of clinically important anti-Vel identified in patient sera.