Investigating Translocation Mechanisms of TDP-43 Into the Endoplasmic Reticulum Using Budding Yeast Models.
Saito Miku M, Tsuzuki Hana H, Hosomi Akira A
Signal peptides direct secretory and membrane proteins to the endoplasmic reticulum (ER), but proteins lacking classical signal peptides can occasionally engage the ER translocation machinery. TDP-43 is a nuclear RNA-binding protein implicated in amyotrophic lateral sclerosis and frontotemporal lobar degeneration. TDP-43 pathology has been linked to extracellular TDP-43 species, association with the ER luminal oxidoreductase PDI, and ER stress-related phenotypes, yet whether TDP-43 fragments can directly access the ER lumen remains unclear. Here, we used budding yeast to examine signal peptide-independent ER entry of TDP-43. C-terminal fragments of TDP-43 acquired N-glycans in ste24Δ cells, whereas full-length TDP-43 showed little detectable ER entry. Endo H digestion confirmed N-glycosylation of the fragments, and a protease protection assay supported ER luminal localization of TDP-43(Δ2-89). ER entry was reduced by sec61-41 and sec66Δ, indicating engagement of a Sec61/Sec66-dependent translocation pathway. Deletion analyses identified opposing sequence elements: residues 320-343 facilitated ER entry, whereas an N-terminal region upstream of the nuclear localization signal suppressed it. TDP-43(Δ2-89) also accessed the secretory pathway, and selected disease-associated variants caused Ire1-dependent growth vulnerability without increasing detectable ER translocation. These findings reveal a Ste24-suppressed route for noncanonical ER entry of TDP-43 fragments.