Abstract
Despite its known role as a secreted neuroprotectant, much of the mesencephalic astrocyte-derived neurotrophic factor (MANF) is retained in the endoplasmic reticulum (ER) of producer cells. There, by unknown mechanisms, MANF plays a role in protein folding homeostasis in complex with the ER-localized Hsp70 chaperone BiP. Here we report that the SAF-A/B, Acinus, and PIAS (SAP) domain of MANF selectively associates with the nucleotide binding domain (NBD) of ADP-bound BiP. In crystal structures the SAP domain engages the cleft between NBD subdomains Ia and IIa, stabilizing the ADP-bound conformation and clashing with the interdomain linker that occupies this site in ATP-bound BiP. MANF inhibits both ADP release from BiP and ATP binding to BiP, and thereby client release. Cells lacking MANF have fewer ER stress-induced BiP-containing high molecular weight complexes. These findings suggest that MANF contributes to protein folding homeostasis as a nucleotide exchange inhibitor that stabilizes certain BiP-client complexes.
Highlights
Despite its known role as a secreted neuroprotectant, much of the mesencephalic astrocytederived neurotrophic factor (MANF) is retained in the endoplasmic reticulum (ER) of producer cells
The findings presented here speak to an important intracellular role for MANF in maintenance of protein-folding homeostasis in the ER that may be independent of its activity as a secreted protein involved in intercellular communication
BiP’s essential role in protein-folding homeostasis in the ER readily explains both the basal activation of the unfolded protein response (UPR) observed in MANF knockout cells and the parallels between the phenotype of mice lacking MANF and mice lacking other components of the ER quality control network[7,36]
Summary
Despite its known role as a secreted neuroprotectant, much of the mesencephalic astrocytederived neurotrophic factor (MANF) is retained in the endoplasmic reticulum (ER) of producer cells. Cells lacking MANF have fewer ER stress-induced BiP-containing high molecular weight complexes These findings suggest that MANF contributes to protein folding homeostasis as a nucleotide exchange inhibitor that stabilizes certain BiP-client complexes. Disruption of MANF gene function leads to enhanced activity of UPR markers in cultured cells[6] and in the tissues of MANF knockout mice[7] and worms[8] Together, these observations hint at MANF’s role in the adaptation of cells to the stress imposed by enhanced levels of unfolded ER proteins. Both BiP and MANF are bipartite proteins (Fig. 2b) To further characterize their physical interaction, we immobilized biotinylated MANF, its N-terminal SAPLIP (Saposin-like protein) or its C-terminal SAP (SAF-A/B, Acinus, and PIAS) domains onto a Bio-layer interferometry (BLI) probe and measured the optical interference signal created by BiP binding. We regard it as unlikely that engagement of MANF as a conventional BiP client made an important contribution to the binding signal
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