The yeast vacuolar H
-ATPase (V-ATPase) of budding yeast (
) is regulated by reversible disassembly. Disassembly inhibits V-ATPase activity under low-glucose conditions by releasing peripheral V
...subcomplexes from membrane-bound V
subcomplexes. V-ATPase reassembly and reactivation requires intervention of the conserved regulator of H
-ATPase of vacuoles and endosomes (RAVE) complex, which binds to cytosolic V
subcomplexes and assists reassembly with integral membrane V
complexes. Consistent with its role, the RAVE complex itself is reversibly recruited to the vacuolar membrane by glucose, but the requirements for its recruitment are not understood. We demonstrate here that RAVE recruitment to the membrane does not require an interaction with V
Glucose-dependent RAVE localization to the vacuolar membrane required only intact V
complexes containing the Vph1 subunit, suggesting that the RAVE-V
interaction is glucose-dependent. We identified a short conserved sequence in the center of the RAVE subunit Rav1 that is essential for the interaction with Vph1
and
Mutations in this region resulted in the temperature- and pH-dependent growth phenotype characteristic of
Δ mutants. However, this region did not account for glucose sensitivity of the Rav1-Vph1 interaction. We quantitated glucose-dependent localization of a GFP-tagged RAVE subunit to the vacuolar membrane in several mutants previously implicated in altering V-ATPase assembly state or glucose-induced assembly. RAVE localization did not correlate with V-ATPase assembly levels reported previously in these mutants, highlighting both the catalytic nature of RAVE's role in V-ATPase assembly and the likelihood of glucose signaling to RAVE independently of V
.
The yeast vacuolar H+-ATPase (V-ATPase) of budding yeast (Saccharomyces cerevisiae) is regulated by reversible disassembly. Disassembly inhibits V-ATPase activity under low-glucose conditions by ...releasing peripheral V1 subcomplexes from membrane-bound Vo subcomplexes. V-ATPase reassembly and reactivation requires intervention of the conserved regulator of H+-ATPase of vacuoles and endosomes (RAVE) complex, which binds to cytosolic V1 subcomplexes and assists reassembly with integral membrane Vo complexes. Consistent with its role, the RAVE complex itself is reversibly recruited to the vacuolar membrane by glucose, but the requirements for its recruitment are not understood. We demonstrate here that RAVE recruitment to the membrane does not require an interaction with V1. Glucose-dependent RAVE localization to the vacuolar membrane required only intact Vo complexes containing the Vph1 subunit, suggesting that the RAVE-Vo interaction is glucose-dependent. We identified a short conserved sequence in the center of the RAVE subunit Rav1 that is essential for the interaction with Vph1 in vivo and in vitro. Mutations in this region resulted in the temperature- and pH-dependent growth phenotype characteristic of ravΔ mutants. However, this region did not account for glucose sensitivity of the Rav1-Vph1 interaction. We quantitated glucose-dependent localization of a GFP-tagged RAVE subunit to the vacuolar membrane in several mutants previously implicated in altering V-ATPase assembly state or glucose-induced assembly. RAVE localization did not correlate with V-ATPase assembly levels reported previously in these mutants, highlighting both the catalytic nature of RAVE's role in V-ATPase assembly and the likelihood of glucose signaling to RAVE independently of V1.
The vacuolar H+-ATPase (V-ATPase) is a highly conserved proton pump responsible for the acidification of intracellular organelles in virtually all eukaryotic cells. V-ATPases are regulated by the ...rapid and reversible disassembly of the peripheral V1 domain from the integral membrane Vo domain, accompanied by release of the V1 C subunit from both domains. Efficient reassembly of V-ATPases requires the Regulator of the H+-ATPase of Vacuoles and Endosomes (RAVE) complex in yeast. Although a number of pairwise interactions between RAVE and V-ATPase subunits have been mapped, the low endogenous levels of the RAVE complex and lethality of constitutive RAV1 overexpression have hindered biochemical characterization of the intact RAVE complex. We describe a novel inducible overexpression system that allows purification of native RAVE and RAVE–V1 complexes. Both purified RAVE and RAVE–V1 contain substoichiometric levels of subunit C. RAVE–V1 binds tightly to expressed subunit C in vitro, but binding of subunit C to RAVE alone is weak. Neither RAVE nor RAVE–V1 interacts with the N-terminal domain of Vo subunit Vph1 in vitro. RAVE–V1 complexes, like isolated V1, have no MgATPase activity, suggesting that RAVE cannot reverse V1 inhibition generated by rotation of subunit H and entrapment of MgADP that occur upon disassembly. However, purified RAVE can accelerate reassembly of V1 carrying a mutant subunit H incapable of inhibition with Vo complexes reconstituted into lipid nanodiscs, consistent with its catalytic activity in vivo. These results provide new insights into the possible order of events in V-ATPase reassembly and the roles of the RAVE complex in each event.
The yeast RAVE (Regulator of H
+
-ATPase of Vacuolar and Endosomal membranes) complex and Rabconnectin-3 complexes of higher eukaryotes regulate acidification of organelles such as lysosomes and ...endosomes by catalyzing V-ATPase assembly. V-ATPases are highly conserved proton pumps consisting of a peripheral V
1
subcomplex that contains the sites of ATP hydrolysis, attached to an integral membrane V
o
subcomplex that forms the transmembrane proton pore. Reversible disassembly of the V-ATPase is a conserved regulatory mechanism that occurs in response to multiple signals, serving to tune ATPase activity and compartment acidification to changing extracellular conditions. Signals such as glucose deprivation can induce release of V
1
from V
o
, which inhibits both ATPase activity and proton transport. Reassembly of V
1
with V
o
restores ATP-driven proton transport, but requires assistance of the RAVE or Rabconnectin-3 complexes. Glucose deprivation triggers V-ATPase disassembly in yeast and is accompanied by binding of RAVE to V
1
subcomplexes. Upon glucose readdition, RAVE catalyzes both recruitment of V
1
to the vacuolar membrane and its reassembly with V
o
. The RAVE complex can be recruited to the vacuolar membrane by glucose in the absence of V
1
subunits, indicating that the interaction between RAVE and the V
o
membrane domain is glucose-sensitive. Yeast RAVE complexes also distinguish between organelle-specific isoforms of the V
o
a-subunit and thus regulate distinct V-ATPase subpopulations. Rabconnectin-3 complexes in higher eukaryotes appear to be functionally equivalent to yeast RAVE. Originally isolated as a two-subunit complex from rat brain, the Rabconnectin-3 complex has regions of homology with yeast RAVE and was shown to interact with V-ATPase subunits and promote endosomal acidification. Current understanding of the structure and function of RAVE and Rabconnectin-3 complexes, their interactions with the V-ATPase, their role in signal-dependent modulation of organelle acidification, and their impact on downstream pathways will be discussed.
Inefficient knock-in of transgene cargos limits the potential of cell-based medicines. In this study, we used a CRISPR nuclease that targets a site within an exon of an essential gene and designed a ...cargo template so that correct knock-in would retain essential gene function while also integrating the transgene(s) of interest. Cells with non-productive insertions and deletions would undergo negative selection. This technology, called SLEEK (SeLection by Essential-gene Exon Knock-in), achieved knock-in efficiencies of more than 90% in clinically relevant cell types without impacting long-term viability or expansion. SLEEK knock-in rates in T cells are more efficient than state-of-the-art TRAC knock-in with AAV6 and surpass more than 90% efficiency even with non-viral DNA cargos. As a clinical application, natural killer cells generated from induced pluripotent stem cells containing SLEEK knock-in of CD16 and mbIL-15 show substantially improved tumor killing and persistence in vivo.
Lysosomes and vacuoles are the most acidic organelles in eukaryotic cells and play a central role in cellular metabolism – serving as a location that coordinates anabolic and catabolic processes. ...This coordination requires the activity of a highly conserved proton pump, the Vacuolar H+‐ATPase (V‐ATPase). Interestingly, both V‐ATPase activity and a cell's metabolic state are dependent on glucose levels. Glucose deprivation induces the V‐ATPase to disassemble into 3 distinct subcomplexes: V1, V1C, and Vo. V1 and V1C are released from Vo at the vacuolar membrane, and this disassembly silences both ATP hydrolysis in V1 and proton transport through Vo. Reassembly occurs rapidly but requires both glucose readdition and a conserved V‐ATPase‐specific assembly factor known as the RAVE (Regulator of the H+‐ATPase of Vacuolar and Endosomes) complex in yeast. The RAVE complex, composed of Rav1, Rav2, and Skp1, interacts with each V‐ATPase subcomplex and, upon glucose readdition, recruits cytosolic V1 and V1C to Vo at the vacuolar membrane to allow V‐ATPase reassembly (Smardon et al., (2015) J. Biol. Chem 290:27511). Although RAVE is essential for efficient V‐ATPase reassembly, how RAVE targets the vacuolar membrane in a glucose‐dependent manner and promotes V‐ATPase reassembly is not understood.
Like the V1 subcomplex and V1C subunit, the RAVE complex is reversibly recruited to the vacuolar membrane in response to glucose. Interestingly, we found that RAVE requires the presence of Vo, but neither V1 nor V1C, for its glucose‐dependent vacuolar localization. We identified a 6‐amino acid, conserved motif within Rav1 that is essential for RAVE's vacuolar localization in vivo. In vitro, deletion of this motif diminishes binding between Rav1 and the cytosolic N‐terminal domain of Vo subunit Vph1. These data suggest that this motif is essential for RAVE to identify Vo subunit Vph1 at the vacuolar membrane, but they do not explain the release of RAVE from the membrane upon glucose deprivation. We seek to determine the signaling mechanism and structural changes involved in RAVE's glucose‐dependent activities. V‐ATPase activity, glycolytic enzymes, the Ras/cAMP pathway, PI(3,5)P2 levels, and cytoskeletal elements have all been implicated in V‐ATPase assembly. We are genetically or chemically silencing the activity of each of these factors and assessing the glucose‐dependent localization of GFP‐tagged RAVE subunits. Initial results indicate that RAVE cycles on and off the vacuolar membrane even in the presence of an assembled but inactive V‐ATPase mutant that is incapable of disassembly. Similar experiments will determine the effects of other factors. Detailed biochemical characterization of the RAVE complex has been thwarted by low expression levels of RAVE subunits. However, we can now express and purify milligram quantities of the RAVE complex alone or bound to V1. This will allow us to identify glucose‐sensitive interactions with RAVE in vitro and to test the hypothesis that RAVE undergoes a glucose‐sensitive conformation change that reversibly exposes the Rav1‐Vph1 binding site. This work addresses the molecular mechanisms governing RAVE‐mediated V‐ATPase reassembly and is essential to understanding the central role of the V‐ATPase in cellular metabolism.
Support or Funding Information
NIH GM127364
This is from the Experimental Biology 2019 Meeting. There is no full text article associated with this published in The FASEB Journal.
Abstract only
V‐ATPases are highly conserved proton pumps that acidify multiple organelles, including lysosomes, endosomes, the late Golgi apparatus, synaptic vesicles, and other regulated secretory ...granules. Precise tuning of the luminal pH of these organelles is critical for function, but the factors governing organelle pH control are not completely understood. V‐ATPases are multisubunit complexes consisting of a peripheral subcomplex (V
1
) that contains sites for ATP hydrolysis and an integral membrane subcomplex (V
o
) that contains the proton pore. The Vo a‐subunit is the largest subunit. It is comprised of an N‐terminal cytosolic domain and a C‐terminal domain that forms part of the proton pore. Most organisms encode multiple isoforms of the V
o
a‐subunit that exhibit organelle‐specific localization. We hypothesize that the N‐terminal (NT) domains of a‐subunit isoforms participate in distinct cellular interactions that are critical for isoform‐specific V‐ATPase localization, activity, and regulation. In yeast cells, there are two organelle‐specific isoforms of the V
o
a‐subunit, Vph1 and Stv1. Vph1‐containing V‐ATPases transit through the secretory pathway en route to the lysosome‐like vacuole. Their activity is regulated by reversible disassembly in response to glucose levels, and they require interaction with the yeast RAVE (regulator of acidification of vacuoles and endosomes) for both their initial biosynthetic assembly and for glucose‐dependent reassembly. The Vph1NT domain binds directly to RAVE and is responsible for glucose‐sensitive interactions with the RAVE complex. In addition, the vacuolar lipid PI(3,5)P2 promotes assembly and activity of Vph1‐containing V‐ATPases. Mutations in Vph1NT compromise PI(3,5)P2‐induced activation, suggesting direct binding to lipid. In contrast, Stv1‐containing V‐ATPases assemble and function in the absence of the RAVE complex, and Stv1NT does not bind to RAVE. However, Stv1NT binds to the Golgi‐enriched lipid PI(4)P, and mutations that abolish PI(4)P binding compromise Golgi retention of Stv1‐containing V‐ATPases. The aNT domains of the four human V
o
a‐subunit isoforms exhibit differential recognition of phosphoinositide lipids. Some human aNT domains interact with the human homologue of the RAVE complex. These data indicate that NT domains of V
o
a‐subunit isoforms encode information for localization and regulation of V‐ATPase activity that help determine organelle pH and protect cells from stress.
Support or Funding Information
NIH R01 GM127364 and NIH R01 GM126020
Vacuolar-type ATPases (V-ATPases) are rotary enzymes that acidify intracellular compartments in eukaryotic cells. These multi-subunit complexes consist of a cytoplasmic V
region that hydrolyzes ATP ...and a membrane-embedded V
region that transports protons. V-ATPase activity is regulated by reversible dissociation of the two regions, with the isolated V
and V
complexes becoming autoinhibited on disassembly and subunit C subsequently detaching from V
. In yeast, assembly of the V
and V
regions is mediated by the regulator of the ATPase of vacuoles and endosomes (RAVE) complex through an unknown mechanism. We used cryogenic-electron microscopy of yeast V-ATPase to determine structures of the intact enzyme, the dissociated but complete V
complex and the V
complex lacking subunit C. On separation, V
undergoes a dramatic conformational rearrangement, with its rotational state becoming incompatible for reassembly with V
. Loss of subunit C allows V
to match the rotational state of V
, suggesting how RAVE could reassemble V
and V
by recruiting subunit C.
In the Firmicutes phylum, GpsB is a membrane associated protein that coordinates peptidoglycan synthesis with cell growth and division. Although GpsB has been studied in several bacteria, the ...structure, function, and interactome of
GpsB is largely uncharacterized. To address this knowledge gap, we solved the crystal structure of the N-terminal domain of
GpsB, which adopts an atypical, asymmetric dimer, and demonstrates major conformational flexibility that can be mapped to a hinge region formed by a three-residue insertion exclusive to
. When this three-residue insertion is excised, its thermal stability increases, and the mutant no longer produces a previously reported lethal phenotype when overexpressed in
. In
, we show that these hinge mutants are less functional and speculate that the conformational flexibility imparted by the hinge region may serve as a dynamic switch to fine-tune the function of the GpsB complex and/or to promote interaction with its various partners. Furthermore, we provide the first biochemical, biophysical, and crystallographic evidence that the N-terminal domain of GpsB binds not only PBP4, but also FtsZ, through a conserved recognition motif located on their C-termini, thus coupling peptidoglycan synthesis to cell division. Taken together, the unique structure of
GpsB and its direct interaction with FtsZ/PBP4 provide deeper insight into the central role of GpsB in
cell division.
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