The revised three-step detour pathway in dolichol biosynthesis is evolutionarily conserved in budding yeast

Area of Science:

• Biochemistry
• Molecular Biology
• Genetics

Background:

• Congenital disorders of glycosylation (CDGs) are linked to defects in dolichol biosynthesis.
• The identification of SRD5A1 and its yeast ortholog DFG10 previously suggested a single-step dolichol synthesis from polyprenol.
• A recent discovery of DHRSX in CDG patients proposed a revised three-step detour pathway for dolichol biosynthesis.

Purpose of the Study:

• To investigate the evolutionary conservation of the proposed three-step detour pathway for dolichol biosynthesis.
• To identify a yeast ortholog for the human DHRSX gene involved in dolichol biosynthesis.
• To functionally characterize the role of the identified yeast ortholog in dolichol production and glycosylation.

Main Methods:

• Bioinformatic analysis to identify yeast orthologs of DHRSX.
• Gene deletion experiments in yeast (Saccharomyces cerevisiae) to assess the function of the identified ortholog (TDA5).
• Phenotypic analysis of yeast mutants, including assessment of dolichol levels, polyprenol accumulation, and glycosylation defects.
• Complementation assays involving the expression of human DHRSX, DFG10, or SRD5A3 in the yeast mutant.

Main Results:

• The gene TDA5 was identified as the functional yeast ortholog of human DHRSX.
• Deletion of TDA5 resulted in significant glycosylation defects, reduced dolichol levels, and accumulation of polyprenol in yeast.
• The observed phenotypes in the TDA5 deletion mutant were rescued by the expression of human DHRSX, but not by DFG10 or SRD5A3.

Conclusions:

• Tda5 performs a conserved function analogous to human DHRSX in yeast dolichol biosynthesis.
• The findings demonstrate the conservation of the three-step detour pathway for dolichol biosynthesis in yeast.
• This study supports a broader eukaryotic framework for dolichol biosynthesis, highlighting the evolutionary importance of this pathway.