title: 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 that 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 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, and Tyr) support an enhanced viability during oxidative stress associated with oxidized Kar2, although these alleles are compromised as an ATPase. We reveal that 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.
authors.affiliation: Department of Molecular Medicine, Cornell University, Ithaca, NY 14853, USA.
authors.forename: Mengni
authors.initials: M
authors.lastname: Xu
authors.affiliation: Department of Molecular Medicine, Cornell University, Ithaca, NY 14853, USA.
authors.forename: Heather M
authors.initials: HM
authors.lastname: Marsh
authors.affiliation: Department of Molecular Medicine, Cornell University, Ithaca, NY 14853, USA. Electronic address: css224@cornell.edu.
authors.forename: Carolyn S
authors.initials: CS
authors.lastname: Sevier
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics* / Q000378:metabolism*; D019943:Amino Acid Substitution; D017124:Conserved Sequence; D003545:Cysteine / Q000235:genetics* / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics* / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development
chemical_list: D005656:Fungal Proteins; D018840:HSP70 Heat-Shock Proteins; C060153:KAR2 protein, yeast; D000251:Adenosine Triphosphatases; D003545:Cysteine
keywords: Hsp70; Kar2; heat shock; oxidative stress; redox signaling