Tuning the Hsp70 chaperone cycle: emerging roles of GrpE-like nucleotide exchange factors in proteostasis and organelle function

The heat shock protein 70 (Hsp70) family of molecular chaperones is essential for nearly every cell to support protein homeostasis through folding, signaling, and quality control. Hsp70 functionality critically depends on co-chaperones, including the GrpE-like family of nucleotide exchange factors (NEFs), first identified in Escherichia coli as GrpE. These factors have long been recognized for their ability to catalyze the release of Hsp70 nucleotide and protein substrates, but recent structural and functional studies have revealed that GrpE-like NEFs are more than passive exchange catalysts, instead acting as dynamic regulators that coordinate chaperone activity with cellular stress responses, organelle-specific demands, and allosteric control of substrate binding and release. In this review, we synthesize decades of research on GrpE-like proteins across bacteria and eukaryotes, culminating in high-resolution structures of the human mitochondrial NEF, GrpEL1, in complex with mitochondrial Hsp70. We examine how architectural features of GrpE-like NEFs have evolved to meet specialized demands, such as thermosensing in bacteria, redox-responsive regulation in vertebrates, and coordination of protein import in mitochondria. We further describe how discrete structural domains dynamically control chaperone cycling, including nucleotide and substrate release, and how gene duplication and domain specialization have driven functional diversification in higher eukaryotes. Finally, we highlight emerging evidence linking NEF activity to mitochondrial homeostasis, stress adaptation, and disease, reframing GrpE-like NEFs as tunable regulators rather than static cofactors. This perspective positions them as stress-adaptive control points in proteostasis and offers a conceptual framework for understanding how ancient chaperone systems have evolved to meet the regulatory needs of modern and complex eukaryotic cells.