Transmembrane Topography of the 100-kDa a Subunit (Vph1p) of the Yeast Vacuolar Proton-translocating ATPase

Abstract

The membrane topography of the yeast vacuolar proton-translocating ATPase a subunit (Vph1p) has been investigated using cysteine-scanning mutagenesis. A Cys-less form of Vph1p lacking the seven endogenous cysteines was constructed and shown to have 80% of wild type activity. Single cysteine residues were introduced at 13 sites within the Cys-less mutant, with 12 mutants showing greater than 70% of wild type activity. To evaluate their disposition with respect to the membrane, vacuoles were treated in the presence or absence of the impermeant sulfhydryl reagent 4-acetamido-4-maleimidylstilbene-2,2-disulfonic acid (AMS) followed by the membrane permeable sulfhydryl reagent 3-(Nmaleimidylpropionyl) biocytin (MPB). Three of the 12 active cysteine mutants were not labeled by MPB. The mutants E3C, D89C, T161C, S266C, N447C, K450C, and S703C were labeled by MPB in an AMS-protectable manner, suggesting a cytoplasmic orientation, whereas G602C and S840C showed minimal protection by AMS, suggesting a lumenal orientation. Factor Xa cleavage sites were introduced at His-499, Leu-560, and Pro-606. Cleavage at 560 was observed in the absence of detergent, suggesting a cytoplasmic orientation for this site. Based on these results, we propose a model of the a subunit containing nine transmembrane segments, with the amino terminus facing the cytoplasm and the carboxyl terminus facing the lumen.The vacuolar proton-translocating ATPases (or V-ATPases) 1 are multisubunit complexes found in a variety of intracellular compartments, such as clathrin-coated vesicles, endosomes, lysosomes, Golgi-derived vesicles, chromaffin granules, synaptic vesicles, and the central vacuoles of yeast, Neurospora, and plants (1-9). Acidification of these intracellular compartments is in turn essential for a variety of cellular processes, including receptor-mediated endocytosis, intracellular targeting, protein processing and degradation, and coupled transport. V-ATPases are also present in the plasma membrane of certain specialized cells, where they function in such processes as renal acidification (7), bone resorption (10), and pH homeostasis (11).The V-ATPases from fungi, plants, and animals are structurally very similar and are composed of two functional domains (1). The V 1 domain is a peripheral complex of molecular mass of 570 kDa composed of eight different subunits of molecular mass 70 -14 kDa (subunits A-H) that is responsible for ATP hydrolysis. The V 0 domain is a 260-kDa integral complex composed of five subunits of molecular mass 100 -17 kDa (subunits a, d, c, cЈ, and cЉ) that is responsible for proton translocation. This structure is similar to that of the ATP synthases (or F-ATPases) that function in ATP synthesis in mitochondria, chloroplasts and bacteria (12-17), and sequence homology between these classes of ATPase has been demonstrated for both the nucleotide binding subunits (A, B, ␣, and ␤) (18, 19) and the proteolipid subunits (subunits c, cЈ, and cЉ) (20,21). No sequence homology has been identified for any of the remaining subun...

Models for the a subunits of the Thermus thermophilus V/A-ATPase and Saccharomyces cerevisiae V-ATPase enzymes by cryo-EM and evolutionary covariance

“…The model of the a subunit presented here is in excellent agreement with these studies. First, the model agrees well with experiments that probed topology and accessibility of the a subunit by labeling residues mutated to cysteine to define them as luminal, membrane-embedded, cytoplasmic, or at the cytoplasmic border (31,38,39) (Fig. 4A).…”

“…Other than these differences, comparison of the two states reveals only minor conformational changes in individual subunits, suggesting little flexibility in the enzyme as a whole (Movie S3). The T. thermophilus V/A-ATPase thus seems to be relatively rigid compared with the S. cerevisiae V-ATPase (8) Numerous studies, many by Forgac and coworkers, have investigated the topology, function, and subunit arrangement of the S. cerevisiae V-ATPase V O region (31,34,(38)(39)(40)(41)(42)(43)(44). The model of the a subunit presented here is in excellent agreement with these studies.…”

N‐terminal domain of the V‐ATPase a2‐subunit displays integral membrane protein properties

“…In the earlier studies, [5][6][7][8] it was difficult to determine the orientation of the N-terminus of a-subunit isoforms, but it is now generally accepted that this domain is oriented toward the cytoplasm. Moreover, according to several recent electron microscopy studies of V-ATPases from different organisms 16,[38][39][40] and small-angle X-ray scattering data of NtpI 1-341 , 26 it was determined that the N-terminal domain of the a-subunit forms an elongated 1-shaped structure.…”

“…1 From a structural perspective, the topology of a-subunit is not understood. 4 For the yeast ''a'' subunit homolog Vph1, several topology models have been proposed over the years, [5][6][7][8] with the most recent model depicting eight transmembrane a-helices with both the Nterminal and C-terminal ends located on the cytoplasmic side of the membrane. 8 Although the location of the N-terminal region of the protein on the cytoplasmic site has been experimentally confirmed, its exact length has not been finally determined.…”

Subunit Interactions in the Clathrin-coated Vesicle Vacuolar (H+)-ATPase Complex

“…Reaction with MBP in the dark followed by irradiation with ultraviolet light gave rise to a 135-kDa product that was recognized by the antibody against subunit E. The size of this complex is consistent with cross-linking of subunit E and subunit a of the V 0 domain. We have recently shown that subunit a contains a large soluble domain at the amino terminus, which is exposed on the cytoplasmic side of the membrane and is therefore situated to interact with the V 1 domain (35).…”