Abstract
Members of a large family of Ankyrin Repeat Domain (ANKRD) proteins regulate numerous cellular processes by binding to specific protein targets and modulating their activity, stability, and other properties. The same ANKRD protein may interact with different targets and regulate distinct cellular pathways. The mechanisms responsible for switches in the ANKRDs' behavior are often unknown. We show that cells' metabolic state can markedly alter interactions of an ANKRD protein with its target and the functional outcomes of this interaction. ANKRD9 facilitates degradation of inosine monophosphate dehydrogenase 2 (IMPDH2), the rate-limiting enzyme in GTP biosynthesis. Under basal conditions ANKRD9 is largely segregated from the cytosolic IMPDH2 in vesicle-like structures. Upon nutrient limitation, ANKRD9 loses its vesicular pattern and assembles with IMPDH2 into rodlike filaments, in which IMPDH2 is stable. Inhibition of IMPDH2 activity with ribavirin favors ANKRD9 binding to IMPDH2 rods. The formation of ANKRD9/IMPDH2 rods is reversed by guanosine, which restores ANKRD9 associations with the vesicle-like structures. The conserved Cys109Cys110 motif in ANKRD9 is required for the vesicle-to-rods transition as well as binding and regulation of IMPDH2. Oppositely to overexpression, ANKRD9 knockdown increases IMPDH2 levels and prevents formation of IMPDH2 rods upon nutrient limitation. Taken together, the results suggest that a guanosine-dependent metabolic switch determines the mode of ANKRD9 action toward IMPDH2.
Highlights
Members of a large family of Ankyrin Repeat Domain (ANKRD) proteins regulate numerous cellular processes by binding to specific protein targets and modulating their activity, stability, and other properties
inosine monophosphate dehydrogenase 2 (IMPDH2) transitions from a diffuse cystolic form to rods when inhibited with ribavirin; this transition is reversed with guanosine. d, HeLa cells were treated with 10 M ribavirin for 1 h transfected with FLAG-ANKRD9 and immunostained for FLAG and IMPDH2
Gray arrows point to rods containing both IMPDH2 and ANKRD9
Summary
This work has started with an observation that expression of a FLAG-tagged ANKRD9 in HeLa cells produced two very different intracellular patterns. Cells were grown under nutrient-limiting conditions for 24 h to induce partial rod formation, washed and placed into the basal growth medium for 24 h This treatment decreased the abundance of rods and caused appearance of ANKRD9 in vesicle-like structures, but did not fully restore the vesicular pattern of ANKRD9 (Fig. S2). The expression levels of ANKRD9 mutants did not differ significantly from the WT (Fig. 7c) These results suggest that Cys109 and Cys110 do not play an important role in stabilizing ANKRD9 in the vesicle-like structures, they are required for assembly with IMPDH2 under nutrient limitation. This finding suggests that ANKRD9 serves to stabilize IMPDH2 rods when nutrients (presumably GTP pools) are depleted (Fig. 8c)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
