The integrated stress response (ISR) is regulated by kinases that phosphorylate the α subunit of translation initiation factor 2 and phosphatases that dephosphorylate it. Genetic and biochemical observations indicate that the eIF2αP-directed holophosphatase, a therapeutic target in diseases of protein misfolding, is comprised of a regulatory subunit, PPP1R15, and a catalytic subunit, protein phosphatase 1 (PP1). In mammals, there are two isoforms of the regulatory subunit, PPP1R15A and PPP1R15B, with overlapping roles in the essential function of eIF2αP dephosphorylation. However, conflicting reports have appeared regarding the requirement for an additional co-factor, G-actin, in enabling substrate-specific dephosphorylation by PPP1R15-containing PP1 holoenzymes. An additional concern relates to the sensitivity of the holoenzyme to the [(o-chlorobenzylidene)amino]guanidines Sephin1 or guanabenz, putative small-molecule proteostasis modulators. It has been suggested that the source and method of purification of the PP1 catalytic subunit and the presence or absence of an N-terminal repeat-containing region in the PPP1R15A regulatory subunit might influence the requirement for G-actin and sensitivity of the holoenzyme to inhibitors. We found that eIF2αP dephosphorylation by PP1 was moderately stimulated by repeat-containing PPP1R15A in an unphysiological low ionic strength buffer, whereas stimulation imparted by the co-presence of PPP1R15A and G-actin was observed under a broad range of conditions, low and physiological ionic strength, regardless of whether the PPP1R15A regulatory subunit had or lacked the N-terminal repeat-containing region and whether it was paired with native PP1 purified from rabbit muscle or recombinant PP1 purified from bacteria. Furthermore, none of the PPP1R15A-containing holophosphatases tested were inhibited by Sephin1 or guanabenz.Supported by a Wellcome Trust Principal Research Fellowship to D.R. (Wellcome 200848/Z/16/Z) and a Wellcome Trust Strategic Award to the Cambridge Institute for Medical Research (Wellcome 100140). M.B. was supported by a Flemish Concerted Research Action (GOA15/016). W.P. was supported by National Institute of Health R01NS091336 and the American Diabetes Association Pathway to Stop Diabetes Grant 1-14-ACN-31. Z.C. is a PhD fellow of the Fund for Scientific Research - Flanders
