substrate translocation into the lumen. The KFERQ pentapeptide, present in ~30% of cytosolic proteins, consists of a glutamine residue (Q) in the first or last position, one of the two positively charged residues lysine (K) or arginine (R), one of the two negatively charged residues glutamic acid (D) or aspartic acid (E) and one of four hydrophobic residues phenylalanine (F), isoleucine (I), leucine (L) or valine (V); the fifth residue can be one of the positive or one of the four hydrophobic residues. The presence of charge in the sequence makes it possible to render an otherwise incomplete motif into a complete one via charge-conferring post-translational modifications such as phosphorylation or acetylation.
Hspa8 is the constitutively expressed cognate heat shock chaperone. Hspa8 is involved in a broad range of functions - from its well-known role in the uncoating of endocytosed clathrin-coated vesicles, to the various aspects of protein homeostasis (folding, assembly, translocation, maturation, degradation such as CMA but also via ubiquitin-proteasome), to regulation of signaling and other cellular processes. Other chaperones, co-chaperones, factors interact with Hspa8 and assist its functions. In CMA, these may include Hsp90aa1 (Hsp90), Dnajb1 (Hsp40), St13 (Hip), Stip1 (Hop), Bag1. Structurally, Hspa8 is composed of a nucleotide binding domain (ATPase) NBD consisting of lobe I and II further subdivided into two subdomains, and a substrate binding domain SBD with a beta-sandwich that binds the client protein and a helix that acts as a lid over it. To see the structure of NBD click here; to see the SBD structure of a close homolog, click here. Hspa8 binds the substrate in the low-affinity, fast-exchange ATP-bound state. Upon ATP hydrolysis, aided by Hsp40, Hspa8 is in the high-affinity, low-exchange ADP-bound state. Exchange of ADP for ATP and going back into the ATP-bound state, aided by several factors referred to as nucleotide exchange factors (NEF) such as Bag1, facilitates substrate release. The inherent flexibility of Hspa8 allows it to interact with many substrates, span a range of conformations and underlies the breadth of its functions. The Lamp2 gene, via alternative splicing, gives rise to several isoforms with different cytosolic and transmembrane regions; only Lamp2a participates in CMA. Once bound to the substrate, Lamp2a undergoes a monomer to multimer transition.
Translocation of the substrate into the lumen of the lysosome requires unfolding, possibly aided by a lysosomal form of Hspa8 whose presence is thought to also be required for translocation. However, the translocation mechanism is poorly understood. Upon substrate translocation, the multimer disassembles for another round of substrate loading. The assembly/disassembly is regulated by multiple factors. Gfap protein binds the translocation complex and stabilizes it. Once the substrate has passed through the complex, Gfap dissociates and binds a phosphorylated Gfap, resident in the lysosomal membrane and associated with Eef1a1, in turn promoting disassembling of the translocation complex. CMA function declines with age and is affected in diseases, generally reduced in neurodegeneration and enhanced in cancer. Impairment of CMA is observed in Parkinson disease (PD) and Alzheimer's (AD). PD associated proteins such as alpha-synuclein (SNCA) or LRRK2 are CMA targets. Pathogenic mutations in these genes affect their processing by CMA. They bind Lamp2a with abnormally high affinity, preventing their translocation into the lumen - click to link to the diagram page for PD pathway. In addition, they also inhibit the degradation of other CMA substrates. Several cathepsins, a main class of lysosomal proteases, are involved in the degradation of monomeric alpha-synuclein and other genes associated with neurodegenerative diseases.To see the ontology for GViewer, annotations and download, click here...(less)