Both Ncs2 and Ncs6 are necessary for the thiolation of U54 in cytoplasmic tRNA. The sulphur group originates from cysteine and flows through an intricate enzymatic cascade of reactions to end up as thiocarboxylated Urm1p (Urm1p-COSH) which is the substrate of 2-thiouridine formation catalysed by the complex Ncs2-Ncs6. While starting with the same cysteine desulfurase Nsf1, the rest of the sulphur relay system is different than in mitochondria. It is definitively distinct from the IscS/Tus-proteins dependent system working in bacteria. In nematode and fission yeast, Cut1-Cut2 complex are the homologs of Ncs6-Ncs2 (Tuc1-Tuc2). At variance with bacterial thiolation system, s2U formation at the wobble position 34 in tRNA can occur only after prior C5-modification of uridine ring. Ncs2 and Ncs6 are down-regulated in a proteasomal dependent fashion in yeast exposed to mild heat stress. This down-regulation influences translation of mRNAs rich in codons decoded by tRNAs thiolated by the Ncs2-Ncs6 complex.