A Conserved Cysteine within the ATPase Domain of the Endoplasmic Reticulum Chaperone BiP is Necessary for a Complete Complement of BiP Activities

Abstract

Among the amino acids, cysteine stands apart based on its highly reactive sulfur group. In general, cysteine is underrepresented in proteins. Yet when present, the features of cysteine often afford unique function. We have shown previously that a cysteine within the ATPase domain of yeast BiP (Kar2) serves as a sensor of the endoplasmic reticulum (ER) redox environment [1, 2]. Under conditions of increased oxidant (oxidative stress) this cysteine becomes oxidized, changing Kar2 from an ATP-dependent foldase to an ATP-independent holdase. We were struck by the high degree of conservation for this cysteine between BiP orthologs, and we sought to determine how cysteine substitution impacts Kar2 function. We observed no single amino acid replacement is capable of recreating the range of functions that can be achieved by wild-type Kar2 with its cysteine in either the unmodified or oxidized states. However, we were able to generate mutants that could selectively replicate the distinct activities exhibited by either unmodified or oxidized Kar2. We found that the ATPase activity displayed by unmodified Kar2 is fully maintained when Cys63 is replaced with Ala or Val. Conversely, we demonstrate that several amino acid substitutions (including His, Phe, Pro, Trp, Tyr) support an enhanced viability during oxidative stress associated with oxidized Kar2, although these alleles are compromised as an ATPase. We reveal the range of activity demonstrated by wild-type Kar2 can be replicated by co-expression of Kar2 mutants that mimic either the unmodified or oxidized Kar2 state, allowing for growth during standard and oxidative stress conditions.

Disrupted Hydrogen-Bond Network and Impaired ATPase Activity in an Hsc70 Cysteine Mutant

“…49 Notably, studies of different amino acid substitutions of the conserved BiP cysteine in yeast revealed also the strongest phenotypes with a BiP C 63 W allele, relative to a BiP C 63 K mutant. 50…”

“…49,55 As mentioned above, these observations are consistent with our prior data relating to BiP, which demonstrated a more severe effect on chaperone activities for a BiP C 63 W mutation than for a C 63 K mutation. 50…”

Redox signaling and unfolded protein response coordinate cell fate decisions under ER stress

“…63p [139]. The ATPase domain of BiP contains a conserved cysteine (Cys63) [140], which can be sulfenylated [138] or glutathionylated [60], suggesting a redox-regulated mechanism of BiP release and UPR initiation. The redox regulation of BiP function is delicate.…”

Diminished Ost3-dependent N-glycosylation of the BiP nucleotide exchange factor Sil1 is an adaptive response to reductive ER stress

“…Residues C52 and C57 of Sil1 have recently been shown to form a redox-active cysteine pair that facilitates the reduction of C63 in the ATPase domain of Kar2 (8). Oxidation of C63 impairs Kar2's ATPase activity, altering its chaperone function to cope with the suboptimal folding conditions that arise during oxidative stress (8)(9)(10)(11). Sil1 can then reduce the oxidized cysteine residue in the BiP ATPase domain to restore ATPase activity and chaperone function once the levels of oxidative stress in the ER have subsided.…”

“…Upon increased ER oxidation, a highly conserved cysteine residue, C63, within the ATPase domain of BiP becomes oxidized (8,9). This alters BiP/Kar2 chaperone activity to limit polypeptide aggregation during suboptimal redox conditions for ER protein folding (9)(10)(11). Reductive stress also gives rise to the accumulation of misfolded proteins.…”