Abstract
Cdc48 is a AAA+ ATPase that plays an essential role for many cellular processes in eukaryotic cells. An archaeal homologue of this highly conserved enzyme was shown to directly interact with the 20S proteasome. Here, we analyze the occurrence and phylogeny of a Cdc48 homologue in Actinobacteria and assess its cellular function and possible interaction with the bacterial proteasome. Our data demonstrate that Cdc48-like protein of actinobacteria (Cpa) forms hexameric rings and that the oligomeric state correlates directly with the ATPase activity. Furthermore, we show that the assembled Cpa rings can physically interact with the 20S core particle. Comparison of the Mycobacterium smegmatis wild-type with a cpa knockout strain under carbon starvation uncovers significant changes in the levels of around 500 proteins. Pathway mapping of the observed pattern of changes identifies ribosomal proteins as a particular hotspot, pointing amongst others toward a role of Cpa in ribosome adaptation during starvation.
Highlights
Energy-dependent chaperones and chaperone-protease complexes comprise important cellular components guarding protein homeostasis in all kingdoms of life
As Cdc48 in eukaryotes and archaea is amongst other functions tied to protein degradation, we set out to investigate if its homolog might have an analogous function in actinobacteria
The only previous study of an actinobacterial Cdc48-like ATPase is for the ortholog from Mycobacterium smegmatis (Msm), and it reports that the protein exists as a monomer in solution as well as in the crystal structure (Unciuleac et al, 2016)
Summary
Energy-dependent chaperones and chaperone-protease complexes comprise important cellular components guarding protein homeostasis in all kingdoms of life. The two non-homologous activators Mpa/ ARC and Bpa both form ring-shaped complexes with a central pore and share as one additional important feature with eukaryotic proteasome interactors a C-terminal proteasome interaction motif featuring a penultimate tyrosine (Delley et al, 2014; Imkamp et al, 2015) In eukaryotes, this motif is called HbYX motif, since the tyrosine is preceded by a hydrophobic residue. The exact role played by Cdc during ERAD is not fully understood, it is involved in pulling proteins from the membrane or from ribosomes during co-translational degradation, and recent evidence suggests even a direct association with the 20S particle (Barthelme and Sauer, 2013) Such a direct role as 20S proteasome activator has been shown for the Cdc homolog from archaea in vitro (Barthelme et al, 2014). We report the comparative proteomic profile of an Msm parent and cdc48-deficient strain, providing a basis for assessing the function of this AAA + ATPase in mycobacteria
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