Required for the efficient 2-thiolation of 5-methyluridine residue at position 54 in the T loop of tRNAs, leading to 5-methyl-2-thiouridine (m(5)s(2)U or s(2)T). TtuD is a sulfur carrier protein that has a role to direct sulfur flow from cysteine desulfurases to m(5)s(2)U synthesis in vivo. It enhances the cysteine desulfurase activity of IscS and SufS, as well as the formation of thiocarboxylated TtuB (TtuB-COSH) in the presence of these desulfurases