When overexpression mimics silencing: mechanistic insights into ubiquitin-like protein conjugation enzymes

Ufmylation is a posttranslational modification, in which the modifier UFM1 is attached to target proteins. Ufmylation is facilitated by a 3-enzyme cascade involving: E1-UBA5, E2-UFC1 and E3-UFL1. Initially, UFM1 is activated by UBA5. Then, UFM1 is transferred from UBA5 to UFC1, forming a thioester bond with the latter. Finally, with the help of UFL1, UFM1 is transferred to the target protein, resulting in UFM1 conjugation. While deletion of each of these enzymes can prevent the conjugation process, how overexpression of these enzymes affects this process is yet not clear. Here, we found that overexpression of UBA5 mimics the effect of its deletion. Both manipulations damage the ability of cells to migrate. At the mechanistic level, we found that UBA5 overexpression reverses the trans-thiolation reaction thereby leading to a back transfer of UFM1 from UFC1 to UBA5. This, similarly to the situation in cells lacking UBA5, reduces the level of charged UFC1 and prevents modifications of target proteins by UFM1. Next, to further understand, we quantified the expression levels of the UFM1 and the UFM1 enzymes in cells. Intriguingly, cells possess the higher level of UFC1 than that of UBA5 and are limited in the amount of free UFM1. Upon UBA5 overexpression, the physiological level of UFM1 does not satisfy the charging of high level expressed UBA5, resulting in the back transfer of UFM1 from UFC1. Overall, our results show that overexpression of an E1 can prevent modification by UFM1.