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The molecular basis of Htm1-mediated commitment of misfolded glycoproteins to endoplasmic reticulum-associated degradation

  • Author(s): Liu, Yi-Chang
  • Advisor(s): Wiessman, Jonathan S
  • Galonic Fujimori, Danica
  • et al.
Abstract

In eukaryotes, nascent proteins that are destined for the secretory pathway enter the endoplasmic reticulum (ER) in unfolded states and generally leave only after they have reached their native conformation. Protein folding in the ER is challenging and often error-prone. To prevent accumulation of misfolded proteins, the ER uses the ER-associated protein degradation (ERAD) machinery to detect and commit these defective proteins for retrotranslocation to cytosol for degradation via the ubiquitin-proteasome system. Previously, our group and others discovered an ERAD pathway that requires the generation of a specific N-glycan structure on misfolded luminal N-glycoproteins to permit their degradation. This N-glycan degradation signal can potentially serve as a mark to flag the defective states of the attached protein, but whether the generation of this N-glycan degradation signal is directly connected to the folding state of the attached protein has remained unknown.

My thesis research work is focused on characterization of Htm1p, the mannosidase that is responsible for generating this N-glycan signal in Saccharomyces cerevisiae. Utilizing an in vitro reconstitution system of Htm1p in complex with its interacting partner, Pdi1p, and glycoprotein substrates whose structures can be closely manipulated, I find that unlike other members of the same mannosidase family, Htm1p-Pdi1p is a glycoprotein-specific mannosidase that has little activity against free glycan. I find that Htm1p-Pdi1p targets intrinsically and artificially misfolded proteins but not their native counterparts. Most importantly, I find that Htm1p-Pdi1p differentiates detailed folding states of misfolded proteins and preferentially acts on partially folded proteins to globally unfolded species. I further find that by forming a tight complex with Pdi1p, Htm1p blocks the intrinsic oxidoreductase function of Pdi1p but keeping the chaperone activity, suggesting a potential mechanism of how the Htm1p-Pdi1p complex recognizes misfolded proteins. In summary, this work demonstrates the tight correlation between the generation of the N-glycan degradation signal and the folding state of the attached protein, opening a future direction to investigate how the ER tells the bad from the good.

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