Functional Redundancy between Two Caenorhabditis elegans Nucleotide Sugar Transporters with a Novel Transport Mechanism

Abstract

Transporters of nucleotide sugars regulate the availability of these substrates required for glycosylation reactions in the lumen of the Golgi apparatus and play an important role in the development of multicellular organisms. Caenorhabditis elegans has seven different sugars in its glycoconjugates, although 18 putative nucleotide sugar transporters are encoded in the genome. Among these, SQV-7, SRF-3, and CO3H5.2 exhibit partially overlapping substrate specificity and expression patterns. We now report evidence of functional redundancy between transporters CO3H5.2 and SRF-3. Reducing the activity of the CO3H5.2 gene product by RNA interference (RNAi) in SRF-3 mutants results in oocyte accumulation and abnormal gonad morphology, whereas comparable RNAi treatment of wild type or RNAi hypersensitive C. elegans strains does not cause detectable defects. We hypothesize this genetic enhancement to be a mechanism to ensure adequate glycoconjugate biosynthesis required for normal tissue development in multicellular organisms. Furthermore, we show that transporters SRF-3 and CO3H5.2, which are closely related in the phylogenetic tree, share a simultaneous and independent substrate transport mechanism that is different from the competitive one previously demonstrated for transporter SQV-7, which shares a lower amino acid sequence identity with CO3H5.2 and SRF-3. Therefore, different mechanisms for transporting multiple nucleotide sugars may have evolved parallel to transporter amino acid divergence.

Glycosylation Genes Expressed in Seam Cells Determine Complex Surface Properties and Bacterial Adhesion to the Cuticle of Caenorhabditis elegans

“…Seven other transporter genes (C53B4.6, F15B10.1, ZK370.7/ugtp-1, ZC250.3, C03H5.2, B0212.4, F54E7.1/pst-2) were tested by RNAi for altered sensitivity to M. nematophilum or other effects, using both rrf-3 and bus-12(br5) mutant backgrounds, but no abnormalities or bacterial resistances were observed. Caffaro et al (2007) have reported gonadal abnormalities associated with RNAi knockdown of C03H5.2, but only in a srf-3 mutant background.…”

“…A precedent for such redundancy has been provided by Caffaro et al (2007), who found that RNAi knockdown of C03H5.2 (encoding a nucleotide sugar transporter related to SRF-3) had no obvious effect in wild-type or RNAi hypersensitive strains, but resulted in gonad abnormality in a srf-3 mutant background.…”

“…However, such transporters probably contribute to multiple glycosylation pathways, and indeed, srf-3 mutants were found to have reductions in both O-linked and N-linked glycoconjugates (Cipollo et al 2004). A notable feature of the C. elegans sugar transporters, previously pointed out by Caffaro et al (2007), is that the number of genes encoding putative transporters (18) appears more than sufficient to transport the seven different sugars in its glycoconjugates, especially as some of these transporters can deal with multiple substrates. Yet the two similar transporters SQV-7 and BUS-12, expressed in overlapping tissues such as the seam cells, have entirely different functions, as demonstrated previously for sqv-7 mutants (Herman and Horvitz 1999) and here for bus-12 mutants, We also find no evidence for redundant functions between the two in sqv-7; bus-12 double mutants.…”

“…Current gene models for C. elegans predict .15 different sugar transporters (Caffaro et al 2007(Caffaro et al , 2008, of which three (srf-3, sqv-7, and bus-12) have now been associated with specific biological functions by means of mutations. Deletion alleles have been generated for five of the other predicted sugar transporter genes [ZK896.9(ok3050), K06H6.3(ok3012), F44C8.7(ok2206), T21B6.5(ok3220), and C50F4.14(ok2860)].…”

Inhibition of Nucleotide Sugar Transport in Trypanosoma brucei Alters Surface Glycosylation

“…cerevisiae cells transformed with empty vector p426GPD or p426GPD-TbNST1-4 were individually grown to an A 600 of ϳ2. Spheroplasts were prepared as previously described (39) using zymolase 100 T (Seikagaku America, Rockville, MD) and Golgi-enriched vesicles were obtained by fractionation of spheroplast suspension with successive centrifugation (40). Nucleotide sugar transport assays were carried out as previously described (39).…”