The twisted gene encodes Drosophila protein O-mannosyltransferase 2 and genetically interacts with the rotated abdomen gene encoding Drosophila protein O-mannosyltransferase 1

Abstract

The family of mammalian O-mannosyltransferases includes two enzymes, POMT1 and POMT2, which are thought to be essential for muscle and neural development. Similar to mammalian organisms, Drosophila has two O-mannosyltransferase genes, rotated abdomen (rt) and DmPOMT2, encoding proteins with high homology to their mammalian counterparts. The previously reported mutant phenotype of the rt gene includes a clockwise rotation of the abdomen and defects in embryonic muscle development. No mutants have been described so far for the DmPOMT2 locus. In this study, we determined that the mutation in the twisted (tw) locus, tw(1), corresponds to a DmPOMT2 mutant. The twisted alleles represent a complementation group of recessive mutations that, similar to the rt mutants, exhibit a clockwise abdomen rotation phenotype. Several tw alleles were isolated in the past; however, none of them was molecularly characterized. We used an expression rescue approach to confirm that tw locus represents DmPOMT2 gene. We found that the tw1 allele represents an amino acid substitution within the conserved PMT domain of DmPOMT2 (TW) protein. Immunostaining experiments revealed that the protein products of both rt and tw genes colocalize within Drosophila cells where they reside in the ER subcellular compartment. In situ hybridization analysis showed that both genes have essentially overlapping patterns of expression throughout most of embryogenesis (stages 8-17), while only the rt transcript is present at early embryonic stages (5 and 6), suggesting its maternal origin. Finally, we analyzed the genetic interactions between rt and tw using several mutant alleles, RNAi, and ectopic expression approaches. Our data suggest that the two Drosophila O-mannosyltransferase genes, rt and tw, have nonredundant functions within the same developmental cascade and that their activities are required simultaneously for possibly the same biochemical process. Our results establish the possibility of using Drosophila as a model system for studying molecular and genetic mechanisms of protein O-mannosylation during development.

Figure 1.

Protein sequence alignment of several protein O-mannosyltransferases from different species. DmPOMT2, fruit fly (Drosophila melanogaster) POMT2; AgPOMT2, mosquito (Anopheles gambiae) POMT2; HsPOMT2, human POMT2; ScPmt2p, yeast (S. cerevisiae) protein O-mannosyltransferase 2; RT, D. melanogaster POMT1 (GenBank accession nos. Q9W5D4, XM_312249, NP_037514, NP_009379, and Q9VTK2, respectively). The aligned conserved PMT domain (solid line) and MIR domains (dashed lines) are indicated. Asterisk indicates the position of T(59) → GS mutation identified in the tw¹ allele. Alignment was performed using CLUSTAL_W algorithm (Thompson et al. 1994) at BCM server (http://searchlauncher.bcm.tmc.edu/multi-align).

Figure 2.

Abdomen rotation phenotype of rt and tw mutants. (A) Wild type; (B) rt^P/rt²; (C) tw¹/tw¹; and (D) tw¹/Df(1)su(s)83. Only females are shown; corresponding males have similar phenotypes (see also Figure 3). Ventral view, anterior is up.

Figure 3.

The tw¹ allele is a hypomorphic mutation in the DmPOMT2 gene. The following numbers of flies were analyzed for corresponding genotypes: tw¹/Y, 102 flies; tw¹/Y; UAS–DmPOMT2/+, 40 flies; tw¹/Y; Act5C–GAL4-17/+, 51 flies; tw¹/Y; UAS–DmPOMT2/+; Act5C–GAL4-17/+, 90 flies; tw¹/tw¹, 72 flies; tw¹/tw¹; UAS–DmPOMT2/+, 39 flies; tw¹/tw¹; Act5C–GAL4-17/+, 84 flies; tw¹/tw¹; UAS–DmPOMT2/+; Act5C–GAL4-17/+, 73 flies; tw¹/Df(1)su(s)83, 65 flies; tw¹/Dp(1;Y)y²sc, 89 flies. Error bars indicate standard deviations.

Figure 4.

Subcellular localization of the TW and RT proteins. (A–C) Immunofluorescent staining for HA-tagged TW protein expressed in the third larva instar salivary gland cells of PDI∷GFP transgenic flies (Bobinnec et al. 2003). (A) TW (red, Cy5); (B) PDI-GFP (green); (C) overlay of the red A and green B channels. The staining reveals TW localization in the ER compartment. (D–F) Double-immunofluorescent staining for HA-tagged TW and LVA proteins expressed in the Drosophila S2 cell culture. (D) LVA (red, Cy3); (E) TW (green, FITC); (F) overlay of the red D and green E channels. The staining indicates TW exclusion from the Golgi compartment. (G–I) Double-immunofluorescent staining for TW (HA-tagged) and RT (MYC-tagged) coexpressed in Drosophila culture cells. (G) TW (red, Cy3); (H) RT (green, FITC); (I) overlay of the red (G) and green (H) channels. The double immunostaining shows colocalization of TW and RT within Drosophila cells. Circular staining around nuclei of S2 cells represents perinuclear ER (Okajima et al. 2005). Bars, 20 μm in C and 6 μm in F and I.

Figure 5.

The pattern of rt and tw expression at different embryonic stages as revealed by in situ hybridization. (A–C) tw expression: A, stage 5; B, stage 14; C, stage 15. (D–F) rt expression: D, stage 5; E, stage 14; F, stage 16. At stage 14, the expression of the genes is elevated in the epidermis (arrows), foregut (open triangles), hindgut (solid triangles), and trachea (asterisk). The presence of the rt mRNA at stage 5 in D suggests maternal contribution of the transcript. Anterior is to the left. B, C, E, and F are dorsolateral views.

Figure 6.

Genetic interactions between tw¹ and rt mutant alleles. (A) The suppression of rt mutant phenotype by the tw¹ allele. The following numbers of flies were analyzed for shown genotypes: males rt^P/rt^P, 73 flies; males tw¹/Y; rt^P/rt^P, 35 flies; females rt^P/rt^P, 30 flies; females tw¹/+; rt^P/rt^P, 83 flies; males rt⁵⁷¹/rt⁵⁷¹, 139 flies; males tw¹/Y; rt⁵⁷¹/rt⁵⁷¹, 143 flies; females rt⁵⁷¹/rt⁵⁷¹, 62 flies; females tw¹/+; rt⁵⁷¹/rt⁵⁷¹, 157 flies; males rt^P/rt⁵⁷¹, 131 flies; males tw¹/Y; rt^P/rt⁵⁷¹, 156 flies; females rt^P/rt⁵⁷¹, 45 flies; females tw¹/+; rt^P/rt⁵⁷¹, 73 flies; males rt^P/rt², 59 flies; males tw¹/Y; rt^P/rt², 67 flies; females rt^P/rt², 50 flies; females tw¹/+; rt^P/rt², 124 flies. Error bars indicate standard deviations. (B) The phenotype of tw¹ homo-/hemizygous mutants is not significantly influenced by the level of rt activity. The following numbers of flies were analyzed for shown genotypes: males −tw¹/Y, 99 flies; tw¹/Y; rt⁵⁷¹/+, 116 flies; tw¹/Y; rt⁵⁷¹/rt⁵⁷¹, 70 flies; females −tw¹/tw¹, 90 flies; tw¹/tw¹; rt⁵⁷¹/+, 102 flies; tw¹/tw¹; rt⁵⁷¹/rt⁵⁷¹, 63 flies. To minimize potential influence of different genetic backgrounds, all these flies were obtained from the same cross: tw¹/Y; rt⁵⁷¹/+ × tw¹/tw¹;rt⁵⁷¹/+. Error bars indicate standard deviations.

Figure 6.

Genetic interactions between tw¹ and rt mutant alleles. (A) The suppression of rt mutant phenotype by the tw¹ allele. The following numbers of flies were analyzed for shown genotypes: males rt^P/rt^P, 73 flies; males tw¹/Y; rt^P/rt^P, 35 flies; females rt^P/rt^P, 30 flies; females tw¹/+; rt^P/rt^P, 83 flies; males rt⁵⁷¹/rt⁵⁷¹, 139 flies; males tw¹/Y; rt⁵⁷¹/rt⁵⁷¹, 143 flies; females rt⁵⁷¹/rt⁵⁷¹, 62 flies; females tw¹/+; rt⁵⁷¹/rt⁵⁷¹, 157 flies; males rt^P/rt⁵⁷¹, 131 flies; males tw¹/Y; rt^P/rt⁵⁷¹, 156 flies; females rt^P/rt⁵⁷¹, 45 flies; females tw¹/+; rt^P/rt⁵⁷¹, 73 flies; males rt^P/rt², 59 flies; males tw¹/Y; rt^P/rt², 67 flies; females rt^P/rt², 50 flies; females tw¹/+; rt^P/rt², 124 flies. Error bars indicate standard deviations. (B) The phenotype of tw¹ homo-/hemizygous mutants is not significantly influenced by the level of rt activity. The following numbers of flies were analyzed for shown genotypes: males −tw¹/Y, 99 flies; tw¹/Y; rt⁵⁷¹/+, 116 flies; tw¹/Y; rt⁵⁷¹/rt⁵⁷¹, 70 flies; females −tw¹/tw¹, 90 flies; tw¹/tw¹; rt⁵⁷¹/+, 102 flies; tw¹/tw¹; rt⁵⁷¹/rt⁵⁷¹, 63 flies. To minimize potential influence of different genetic backgrounds, all these flies were obtained from the same cross: tw¹/Y; rt⁵⁷¹/+ × tw¹/tw¹;rt⁵⁷¹/+. Error bars indicate standard deviations.

Figure 7.

Genetic interactions between tw and rt. (A) The increased level of tw does not have a significant effect on the phenotype of rt mutants. The following numbers of flies were analyzed for indicated genotypes: males rt^P/rt⁵⁷¹, 58 flies; males +/Dp(1;Y)y²sc; rt^P/rt⁵⁷¹, 58 flies; males Act5C–GAL4-25/+; rt^P/rt², 55 flies; males Act5C–GAL4-25/UAS–DmPOMT2; rt^P/rt², 43 flies; females Act5C–GAL4-25/+; rt^P/rt², 78 flies; females Act5C–GAL4-25/UAS–DmPOMT2; rt^P/rt², 28 flies. Error bars indicate standard errors of the mean. The difference between phenotypes of compared male genotypes is small but statistically significant (t-test, P < 0.05). The difference between corresponding females is not statistically significant (P > 0.3). (B) The phenotype of rt mutants is insensitive to the decreased level of tw. The following numbers of flies were analyzed for indicated genotypes: males Act5C–GAL4-25/UAS–twRNAi-39, 138 flies; females Act5C–GAL4-25/UAS–twRNAi-39, 111 flies; males Act5C–GAL4-25/+; rt^P/rt², 38 flies; males Act5C–GAL4-25/UAS–twRNAi-39; rt^P/rt², 48 flies; females Act5C–GAL4-25/+; rt^P/rt², 49 flies; females Act5C–GAL4-25/UAS–twRNAi-39; rt^P/rt², 60 flies. The difference between phenotypes of compared genotypes is not statistically significant (t-test, P > 0.07). Error bars indicate standard errors of the mean.

Figure 7.

Genetic interactions between tw and rt. (A) The increased level of tw does not have a significant effect on the phenotype of rt mutants. The following numbers of flies were analyzed for indicated genotypes: males rt^P/rt⁵⁷¹, 58 flies; males +/Dp(1;Y)y²sc; rt^P/rt⁵⁷¹, 58 flies; males Act5C–GAL4-25/+; rt^P/rt², 55 flies; males Act5C–GAL4-25/UAS–DmPOMT2; rt^P/rt², 43 flies; females Act5C–GAL4-25/+; rt^P/rt², 78 flies; females Act5C–GAL4-25/UAS–DmPOMT2; rt^P/rt², 28 flies. Error bars indicate standard errors of the mean. The difference between phenotypes of compared male genotypes is small but statistically significant (t-test, P < 0.05). The difference between corresponding females is not statistically significant (P > 0.3). (B) The phenotype of rt mutants is insensitive to the decreased level of tw. The following numbers of flies were analyzed for indicated genotypes: males Act5C–GAL4-25/UAS–twRNAi-39, 138 flies; females Act5C–GAL4-25/UAS–twRNAi-39, 111 flies; males Act5C–GAL4-25/+; rt^P/rt², 38 flies; males Act5C–GAL4-25/UAS–twRNAi-39; rt^P/rt², 48 flies; females Act5C–GAL4-25/+; rt^P/rt², 49 flies; females Act5C–GAL4-25/UAS–twRNAi-39; rt^P/rt², 60 flies. The difference between phenotypes of compared genotypes is not statistically significant (t-test, P > 0.07). Error bars indicate standard errors of the mean.

Figure 8.

Synergistic genetic interactions between rt and tw. The following numbers of flies were analyzed for indicated genotypes: females Act5C–GAL4-25/+; UAS–twRNAi-77/+, 33 flies; females Act5C–GAL4-25/+; UAS–twRNAi-77/rt^P, 42 flies; males UAS–twRNAi-39/+; tubP–GAL4/+, 198 flies; males UAS–twRNAi-39/+; tubP–GAL4/rt^P, 110 flies; females UAS–twRNAi-39/+; tubP–GAL4/+, 192 flies; females UAS–twRNAi-39/+; tubP–GAL4/rt^P, 84 flies. The difference between compared phenotypes is statistically significant (t-test, P ≪ 0.001). Error bars indicate standard errors of the mean.