ORMDL proteins are a conserved new family of endoplasmic reticulum membrane proteins

Abstract

Background: Annotations of completely sequenced genomes reveal that nearly half of the genes identified are of unknown function, and that some belong to uncharacterized gene families. To help resolve such issues, information can be obtained from the comparative analysis of homologous genes in model organisms.

Results: While characterizing genes from the retinitis pigmentosa locus RP26 at 2q31-q33, we have identified a new gene, ORMDL1, that belongs to a novel gene family comprising three genes in humans (ORMDL1, ORMDL2 and ORMDL3), and homologs in yeast, microsporidia, plants, Drosophila, urochordates and vertebrates. The human genes are expressed ubiquitously in adult and fetal tissues. The Drosophila ORMDL homolog is also expressed throughout embryonic and larval stages, particularly in ectodermally derived tissues. The ORMDL genes encode transmembrane proteins anchored in the endoplasmic reticulum (ER). Double knockout of the two Saccharomyces cerevisiae homologs leads to decreased growth rate and greater sensitivity to tunicamycin and dithiothreitol. Yeast mutants can be rescued by human ORMDL homologs.

Conclusions: From protein sequence comparisons we have defined a novel gene family, not previously recognized because of the absence of a characterized functional signature. The sequence conservation of this family from yeast to vertebrates, the maintenance of duplicate copies in different lineages, the ubiquitous pattern of expression in human and Drosophila, the partial functional redundancy of the yeast homologs and phenotypic rescue by the human homologs, strongly support functional conservation. Subcellular localization and the response of yeast mutants to specific agents point to the involvement of ORMDL in protein folding in the ER.

Figure 1

Nucleotide sequence of the ORMDL1 cDNA. The translation is shown below. Intron positions are marked with black triangles. The exon shown between square brackets in the 5'-UTR is alternatively spliced. Underlines mark the positions of the primers used for the RACE experiments.

Figure 2

Alignment of deduced ORMDL amino-acid sequences. Highly conserved positions (≥ 95%) are shown against a black background, whereas those with conservative exchanges are shown against a grey background. Alignment was performed using the CLUSTALW program [23]. Potential transmembrane segments (TM1 to TM4) are marked with bars above the alignment: Upper bars according to HMMTOP [25] and lower bars following TMAP [26]. Species abbreviations are as follows: Hsap, human; Mmus, mouse; Rnor, rat; Sscr, pig; Btau, cow; Ggal, chicken; Xlae, Xenopus laevis; Stro, Silurana tropicalis; Frub, Takifugu rubripes (pufferfish); Drer, Danio rerio (zebrafish); Cint, Ciona intestinalis; Dmel, Drosophila melanogaster; Atha, Arabidopsis thaliana; Hvul, Hordeum vulgare (barley); Sbic, Sorghum vulgare; Sreb, Stevia rebaudiana; Lesc, Lycopersicon esculentum (tomato); Gmax, Glycine max (soybean); Mtru, Medicago truncatula; Lpen, Lycopersicon pennellii; Zmay, Zea mays (maize); Scer, Saccharomyces cerevisiae; Smon, Saccharomyces monacensis; Spom, Schizosaccharomyces pombe; Ecun, Encephalitozoon cuniculi.

Figure 3

Relationships between the ORMDL amino-acid sequences shown as an unrooted phylogenetic tree. The tree was obtained with the program CLUSTALW [23], with distances corrected for multiple substitutions, and positions with gaps excluded. Numbers show results from bootstrap analysis [4]. The branches are labeled in the same manner as for the alignment (Figure 2).

Figure 4

Gene organization of human ORMDL1, ORMDL2, ORMDL3, ORMDL1, ORMDL2, and Drosophila ORMDL. Exon numbers of ORMDL1 are shown above the bars. Coding exons are shown in color and their sizes (in nucleotides) are: exon 3, 181 (orange); exon 4, 152 (yellow); exon 5, 133 (green). The intron sizes in kilobases are shown in-between the bars. The numbers below the bars denote the amino-acid positions for the exon-intron boundaries, and for the end of the ORF. The dotted appearance of the bar representing exon 2 of ORMDL1 indicates that it is alternatively spliced. The structure of the two human pseudogenes is also shown. The chromosomal location of each gene or pseudogene is indicated to the right.

Figure 5

RT-PCR analyses of expression patterns of ORMDL1, ORMDL2, and ORMDL3. (a) Pattern of expression in a normalized panel of adult and fetal human tissues. (b) Northern blot hybridization using a ORMDL1 probe (containing the whole coding region) against the mRNA of human adult tissues. The size of the three detected transcripts is shown. The same pattern was obtained using a probe containing the ORMDL1 3'-UTR (data not shown).

Figure 6

ORMDL fused to EGFP at the amino or carboxyl terminus localizes in the perinuclear ER and throughout the ER network. (a-c) Confocal scanning microscopy of COS-7 cells transfected with ORMDL1-EGFP (a) co-localize with protein-disulfide isomerase (PDI) (b), an ER-marker protein, showing an overlapping signal in the endoplasmic reticulum (c). (d-f) Double-label fluorescence with biotin-labeled concanavalin A showed no overlapping signal with ORMDL1-EGFP at the plasma membrane. (g-l) COS-7 cells transfected with EGFP-ORMDL1, EGFP-ORMDL2 and EGFP-ORMDL3 fusion proteins were similar either in fixed (g, h, and i, respectively) and in vivo (j) cells, co-localizing with an in vivo ER marker (k). EGFP alone was uniformly distributed throughout the cell, including the nucleus (l).

Figure 7

Whole-mount in situ hybridization to detect DORMDL expression in Drosophila embryos and imaginal discs. DORMDL is ubiquitously expressed in the ectodermal tissues. (a) Syncytial blastoderm (stage 3/4, approximately 2 h); (b) cellular blastoderm focused in the plane where polar cells are visible (stage 5, 3 h-3 h 15 min); (c) lateral view of an stage 11 embryo with the fully stretched germ band; (d) lateral view of an stage 11-12 embryo when the germ band begins to recede; (e) lateral view of a stage 14 embryo; (f) sense probe (stage 13). A, anterior; P, posterior; D, dorsal; V, ventral; pc, polar cells; cel, cellularization; pro, procephalon; gb, germ band; T1-T3, thoracic segments; A1-A9, abdominal segments. Timings of developmental stages are approximate (development at 25°C). (g) Eye-antenna imaginal disc; (h) leg imaginal disc; (i) wing imaginal disc; (j) negative control - wing imaginal disc hybridized with the sense probe.

Figure 8

Qualitative phenotypic analysis of yeast knockout strains and functional complementation of human ORMDL3. (a) Genotype PCR analyses of spores derived from tetrad dissection. The deduced genotype for each clone is indicated above. Four different PCR reactions were performed to assess the following: lane 1, ORM1 integrity; lane 2, ORM1 deletion; lane 3, ORM2 integrity; lane 4, ORM2 deletion. (b) Dropout plate showing the growth of haploid wild-type, single-knockout and double-knockout strains. (c) Growth of wild-type, single-knockout and double-knockout strains as well as functional complementation of double knockouts transformed with human ORMDL3. pSC16-hORMDL3 and pVT-U-hORMDL3 denote, respectively, yeast centromeric and non-centromeric plasmid constructs used in the complementation assay. Five sequential dilutions (1:5 each) were plated, left to right, in YPAD medium supplemented with the toxic agents at the indicated concentrations.