Structural basis of V-ATPase V _O region assembly by Vma12p, 21p, and 22p

Abstract

Vacuolar-type adenosine triphosphatases (V-ATPases) are rotary proton pumps that acidify specific intracellular compartments in almost all eukaryotic cells. These multi-subunit enzymes consist of a soluble catalytic V 1 region and a membrane-embedded proton-translocating V O region. V O is assembled in the endoplasmic reticulum (ER) membrane, and V 1 is assembled in the cytosol. However, V 1 binds V O only after V O is transported to the Golgi membrane, thereby preventing acidification of the ER. We isolated V O complexes and subcomplexes from Saccharomyces cerevisiae bound to V-ATPase assembly factors Vma12p, Vma21p, and Vma22p. Electron cryomicroscopy shows how the Vma12-22p complex recruits subunits a, e, and f to the rotor ring of V O while blocking premature binding of V 1 . Vma21p, which contains an ER-retrieval motif, binds the V O :Vma12-22p complex, “mature” V O , and a complex that appears to contain a ring of loosely packed rotor subunits and the proteins YAR027W and YAR028W. The structures suggest that Vma21p binds assembly intermediates that contain a rotor ring and that activation of proton pumping following assembly of V 1 with V O removes Vma21p, allowing V-ATPase to remain in the Golgi. Together, these structures show how Vma12-22p and Vma21p function in V-ATPase assembly and quality control, ensuring the enzyme acidifies only its intended cellular targets.

ATP6V1A is required for synaptic rearrangements and plasticity in murine hippocampal neurons

“…The loss of expression of Atp6v1a is paralleled by reduction of its main partners in the cytosolic V 1 domain, whereas no similar decrease is detected for Atp6v0a1, a member of the membrane embedded V 0 domain. These findings are consistent with the reported independent biosynthetic origin of V 0 and V 1 subunits 43 and demonstrate an overall decrease of the cytosolic enzymatic V 1 subunit in Atp6v1a-silenced neurons. The closely similar phenotypes which were observed by molecular silencing of Atp6v1a and pharmacological v-ATPase inhibition by bafilomycin, further demonstrate the pilot role of V 1 expression for the physiological regulation of v-ATPase activity that propells both lysosomal acidification and autophagy flux.…”

Identification of a muscle-specific isoform of VMA21 as a potent actor in X-linked myopathy with excessive autophagy pathogenesis

“…Whereas previous reports suggest that VMA21 interacts with the V0c” sub-unit of the v-ATPase [ 6 ], recent cryoEM data demonstrated that yeast Vma21p rather interacts with c sub-units of the V0 domain [ 10 ]. As VMA21-101 and -120 were both detected in the ER/SR of muscle cells, we tested their capacity to bind V0c/c” sub-units.…”

“…The VMA21-101 and VMA21-120 isoforms differ in their N-terminal sequences, which do not contain functionally identified domains. These distinct regions may constitute flexible cytosolic domains that can modulate the binding affinity of VMA21’s two transmembrane helices to the different V0 domain sub-units [ 10 ]. Of note, concomitant to high VMA21-120 levels, VMA21-101 expression was relatively low in skeletal muscle compared to non-muscle tissues.…”

“…It is even more intriguing given that mutations in CCDC115 and TMEM199 , which encode the human homolog of yeast Vma22p and Vma12p, respectively, also lead to CDG predominantly affecting liver [ 42 , 43 ]. CryoEM analysis has placed the chaperones CCDC115 and TMEM199 upstream of VMA21 in v-ATPase assembly [ 10 ]. Similarly, diseases associated with mutations in ATP6AP1 and ATP6AP2 , encoding two central v-ATPase accessory proteins, are characterized by liver dysfunction, with immunodeficiency and cognitive impairment [ 44 , 45 ].…”