Profiling Cysteine Reactivity and Oxidation in the Endoplasmic Reticulum

The
endoplasmic reticulum (ER) is the initial site of biogenesis
of secretory pathway proteins, including proteins localized to the
ER, Golgi, lysosomes, intracellular vesicles, plasma membrane, and
extracellular compartments. Proteins within the secretory pathway
contain a high abundance of disulfide bonds to protect against the
oxidative extracellular environment. These disulfide bonds are typically
formed within the ER by a variety of oxidoreductases, including members
of the protein disulfide isomerase (PDI) family. Here, we establish
chemoproteomic platforms to identify oxidized and reduced cysteine
residues within the ER. Subcellular fractionation methods were utilized
to enrich for the ER and significantly enhance the coverage of ER-localized
cysteine residues. Reactive-cysteine profiling ranked ∼900
secretory pathway cysteines by reactivity with an iodoacetamide–alkyne
probe, revealing functional cysteines annotated to participate in
disulfide bonds, or S-palmitoylation sites within
proteins. Through application of a variation of the OxICAT protocol
for quantifying cysteine oxidation, the percentages of oxidation for
each of ∼700 ER-localized cysteines were calculated. Lastly,
perturbation of ER function, through chemical induction of ER stress,
was used to investigate the effect of initiation of the unfolded protein
response (UPR) on ER-localized cysteine oxidation. Together, these
studies establish a platform for identifying reactive and functional
cysteine residues on proteins within the secretory pathway as well
as for interrogating the effects of diverse cellular stresses on ER-localized
cysteine oxidation.