Abstract
Synthesis of extracellular sulfated molecules requires active 3'-phosphoadenosine 5'-phosphosulfate (PAPS). For sulfation to occur, PAPS must pass through the Golgi membrane, which is facilitated by Golgi-resident PAPS transporters. Caenorhabditis elegans PAPS transporters are encoded by two genes, pst-1 and pst-2. Using the yeast heterologous expression system, we characterized PST-1 and PST-2 as PAPS transporters. We created deletion mutants to study the importance of PAPS transporter activity. The pst-1 deletion mutant exhibited defects in cuticle formation, post-embryonic seam cell development, vulval morphogenesis, cell migration, and embryogenesis. The pst-2 mutant exhibited a wild-type phenotype. The defects observed in the pst-1 mutant could be rescued by transgenic expression of pst-1 and hPAPST1 but not pst-2 or hPAPST2. Moreover, the phenotype of a pst-1;pst-2 double mutant were similar to those of the pst-1 single mutant, except that larval cuticle formation was more severely defected. Disaccharide analysis revealed that heparan sulfate from these mutants was undersulfated. Gene expression reporter analysis revealed that these PAPS transporters exhibited different tissue distributions and subcellular localizations. These data suggest that pst-1 and pst-2 play different physiological roles in heparan sulfate modification and development.
Highlights
The levels of ⌬HexA␣1– 4GlcNAc(6S) were not affected in the pst-1 mutant worms, but they were reduced in the pst-2 mutant worms compared with controls. These results suggest that sulfation patterns of HS depend on both PST-1 and PST-2 activity
The results suggested that pst-1 is involved in post-embryonic seam cell development
PST-1 Expression in the Epidermis Is Sufficient for Proper Larval Epithelial Development, and Its Expression in the Neuroectoblast Is Sufficient for Rescuing Embryonic Lethality—To determine which cell types required pst-1 gene function, we examined whether epidermal, neuronal, or muscle expression of pst-1 was sufficient to rescue the embryonic, vulval, and cuticle phenotypes of deletion mutants
Summary
Materials—[35S]PAPS (1.59 Ci/mmol), GDP-[U-14C]fucose (271 mCi/mmol), and CMP-[9-3H]sialic acid (33.6 Ci/mmol) were purchased from PerkinElmer Life Sciences. The pst-1(tm3364) mutant contained an 1186-bp deletion and a 9-bp insertion, which removed the third and fourth exons of the gene and resulted in a frameshift mutation. The pst-2(tm3316) mutant contained a 296-bp deletion that removed part of the fifth exon, resulting in a frameshift mutation This deletion mutant expressed only five transmembrane regions in the N terminus of the protein. To make the tissue-specific pst-1b::egfp and pst-2a::egfp expression constructs, Pdpy-7::pst-1a::egfp and Pdpy-7::pst-2a::egfp were digested with NotI to yield the pst-1a and pst-2a sequences, respectively These were cloned into Pmyo-3::egfp, Punc-119::egfp, and Prgef-1::egfp plasmids. Each YEp352GAP-II-pst-1a-HA and YEp352GAP-II-pst-2-HA plasmid was transformed into yeast (Saccharomyces cerevisiae) strain W303-1a (MATa, ade, ura, his, trp, leu112, and can1–100) by the lithium acetate procedure. The extent of co-localization of PST-11⁄7EGFP and PST-21⁄7EGFP with AMAN21⁄7mCherry was determined as the percent intensity of the co-localized signal relative to the total GFP-specific signal using the colocalization threshold plug-in within NCBI Image J software
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