scholarly journals Functional significance of the beta-subunit for heterodimeric P-type ATPases.

1995 ◽  
Vol 198 (1) ◽  
pp. 1-17 ◽  
Author(s):  
D C Chow ◽  
J G Forte
Keyword(s):  
Cation Exchange ◽  
Intracellular Trafficking ◽  
Disulfide Bonds ◽  
Basolateral Membrane ◽  
Extracellular Domain ◽  
Beta Subunit ◽  
Alpha Subunit ◽  
Alpha Beta ◽  
The Stability ◽  
P Type

We have reviewed the structural and functional role of the beta-subunit in a subfamily of the P-ATPases known as the alpha/beta-heterodimeric, cation-exchange ATPases. The subfamily consists of the various isoforms of Na+/K(+)-ATPase and H+/K(+)-ATPase, both of which pump a cation out of the cell (Na+ or H+, respectively) in recycle exchange for K+. Much of the earlier work has emphasized the functional activities of the alpha-subunit, which shares many characteristics with the broader P-ATPase family. It is now clear that the glycosylated beta-subunit is an essential component of the cation-exchange ATPase subfamily. All beta-subunit isoforms have three highly conserved disulfide bonds within the extracellular domain that serve to stabilize the alpha-subunit, alpha/beta interaction and functional activity of the holoenzyme. Evidence strongly suggests that the beta-subunit is involved in the K(+)-dependent reactions of the enzymes, such as the E1-E2 transition and K+ occlusion, and that the extracellular domain of the beta-subunit plays an important role in determining the kinetics of K+ interaction. In most vertebrate cells, the unassociated alpha-subunit is restricted to the endoplasmic reticulum (ER), and assembly of the alpha/beta complex occurs within the ER. Signals for exiting the ER and directing the correct intracellular trafficking are primarily determined by the beta-subunit; Na+/K(+)-ATPase typically terminates in the plasma membrane facing the basolateral membrane, whereas all isoforms of H+/K(+)-ATPase terminate in the apical membrane. The C-terminal extracellular domain of the beta-subunit is important for proper interaction with the alpha-subunit and for correct intracellular trafficking. Oligosaccharides on the beta-subunit are not essential for enzyme function, but do serve to enhance the efficiency of alpha/beta association by increasing the lifetime of the unassociated beta-subunit and the stability of the alpha/beta complex to tryptic attack. We propose that highly specialized glycosylation on the beta-subunit of the gastric H+/K(+)-ATPase may help to protect that enzyme from the harsh extracellular environment of the stomach.

Mercury weakens membrane anchoring of Na-K-ATPase

1992 ◽  
Vol 262 (5) ◽  
pp. F837-F842 ◽  
Author(s):  
E. Imesch ◽  
M. Moosmayer ◽  
B. M. Anner
Keyword(s):  
Atpase Activity ◽  
Model Compound ◽  
Beta Subunit ◽  
Alpha Subunit ◽  
Structural Effects ◽  
Subunit Interaction ◽  
Alpha Beta ◽  
Membrane Anchoring ◽  
Atpase Inhibition

The presence of circulating inhibitors able to decrease the renal Na-K-adenosinetriphosphatase (ATPase) activity (natriuretic hormones) was postulated some 30 years ago. In the present work, the natriuretic inhibitor HgCl2 was selected as a model compound for the structural characterization of a possible natriuretic pathway for Na-K-ATPase modification. The structural effects of Na-K-ATPase inhibition by HgCl2 were assessed by trypsinolysis of the blocked enzyme in comparison with untreated preparations. The results show that inactivation of Na-K-ATPase by HgCl2 leads to the release of the alpha-subunit from the membrane preferentially in the E2 conformation but also in the E1 conformation. Apparently, HgCl2 weakens the membrane anchoring of the alpha-subunit, presumably by loosening the alpha-beta-subunit interaction. By this mechanism, the sensitivity of the Na-K-ATPase to extracellular drugs, hormones, and antibodies, as well as to intracellular proteases and other regulatory factors, could be altered.

Analysis of subunit assembly of the Na-K-ATPase

1994 ◽  
Vol 266 (3) ◽  
pp. C579-C589 ◽  
Author(s):  
D. M. Fambrough ◽  
M. V. Lemas ◽  
M. Hamrick ◽  
M. Emerick ◽  
K. J. Renaud ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Sodium Pump ◽  
Beta Subunit ◽  
Alpha Subunit ◽  
Beta Subunits ◽  
Limited Region ◽  
Subunit Assembly ◽  
Alpha Subunits ◽  
Subunit Isoforms ◽  
P Type

The Na-K-ATPase, or sodium pump, is comprised of two subunits, alpha and beta. Each subunit spans the lipid bilayer of the cell membrane. This review summarizes our efforts to determine how the two subunits interact to form the functional ion transporter. Our major approach has been to observe the potential for subunit assembly when one or both subunits are truncated or present as chimeras that retain only a limited region of the Na-K-ATPase. DNAs encoding these altered subunit forms of the avian Na-K-ATPase are expressed in mammalian cells. Monoclonal antibodies specific for the avian beta-subunit are then used to purify newly synthesized avian beta-subunits, and the presence of accompanying alpha-subunits indicates that subunit assembly has occurred. The ectodomain of the beta-subunit (approximately residues 62-304) is sufficient for assembly with the alpha-subunit, and a COOH-terminal truncation of the beta-subunit that lacks aminoacyl residues beyond 162 will assemble inefficiently. A maximum of 26 aminoacyl residues of the alpha-subunit are necessary for robust assembly with the beta-subunit, when this sequence replaces the COOH-terminal half of the loop between membrane spans 7 and 8 in the SERCA1 Ca-ATPase. This region of the Ca-ATPase faces the lumen of the endoplasmic reticulum. These findings encourage study of other related questions, including whether there is preferential assembly of certain subunit isoforms and how various P-type ATPases are targeted to their appropriate subcellular compartments.

scholarly journals Expression of rat endopeptidase-24.18 in COS-1 cells: membrane topology and activity

1994 ◽  
Vol 300 (1) ◽  
pp. 37-43 ◽  
Author(s):  
P E Milhiet ◽  
D Corbeil ◽  
V Simon ◽  
A J Kenny ◽  
P Crine ◽  
...  
Keyword(s):  
Cell Surface ◽  
Culture Media ◽  
Enzymic Activity ◽  
Beta Subunit ◽  
Membrane Topology ◽  
Site Directed Mutagenesis ◽  
Alpha Subunit ◽  
Cell Extracts ◽  
Membrane Bound ◽  
Alpha Beta

Endopeptidase-24.18 (E-24.18; EC 3.4.24.18) is a metallopeptidase of the astacin family and is highly expressed in kidney brush-border membranes of rodents. Rat E-24.18 consists of two disulphide-linked alpha/beta dimers [(alpha/beta)2]. In order to investigate the mechanisms of assembly and the importance of each subunit in the enzymic process, the cloned cDNAs for the rat alpha and beta subunits were transiently expressed either alone or together in COS-1 cells. Immunoblotting of cell extracts and spent culture media showed that, when expressed alone, the alpha subunit is secreted, whereas the beta subunit is membrane-bound. In alpha/beta-transfected cells, the alpha subunit remained membrane-bound, but could be released from the cell surface after papain treatment or after incubation with 10 mM dithiothreitol. Furthermore, mutants of the alpha subunit in which the putative C-terminal anchor domain was deleted could still form cell-associated alpha/beta dimers. These results are consistent with a topological model of E-24.18 in which the beta subunit is anchored in the plasma membrane and the alpha subunit is retained at the cell surface through disulphide bridge(s) with the beta subunit. Both the alpha and beta recombinant subunits expressed in COS-1 cells showed little azocasein-degrading activity. However, activity of either individual subunits of alpha/beta dimers was increased after mild trypsin digestion, suggesting that in COS-1 cells the enzymes are synthesized as zymogens. Finally, inactivation of the alpha subunit by site-directed mutagenesis of Glu-157, which is believed to play a role in catalysis, showed that both subunits participate in the enzymic activity of the heterodimer.

Synthesis and translocation of Na(+)-K(+)-ATPase alpha- and beta-subunits to plasma membrane in MDCK cells

1992 ◽  
Vol 262 (2) ◽  
pp. C470-C483 ◽  
Author(s):  
A. K. Mircheff ◽  
J. W. Bowen ◽  
S. C. Yiu ◽  
A. A. McDonough
Keyword(s):  
Plasma Membrane ◽  
Mdck Cells ◽  
Subcellular Fractionation ◽  
Beta Subunit ◽  
Alpha Subunit ◽  
Beta Subunits ◽  
Madin Darby Canine Kidney ◽  
Intracellular Membranes ◽  
Alpha Beta ◽  
Darby Canine Kidney

Synthesis and translocation of Na(+)-K(+)-ATPase alpha-catalytic and beta-glycoprotein subunits from intracellular membranes to the plasma membrane were studied in Madin-Darby canine kidney cells (MDCK-T) by combining the methods of pulse-chase labeling, subcellular fractionation on sorbitol gradients, and immunoprecipitation. Immunoprecipitation from homogenates revealed that radioactive methionine incorporated into beta-subunit was equal to that incorporated into alpha-subunit after 15 min of labeling. Because the ratio of total methionines in alpha- vs. beta-subunit is approximately 5:1, these results suggest that beta-subunit is synthesized in molar excess over alpha-subunit. Half of the newly synthesized beta-subunit, likely unassembled units, were degraded by 60 min after labeling, while alpha-subunits were stable through 120 min after synthesis, suggesting alpha may be limiting for alpha beta-assembly. By 120 min the ratio of counts incorporated into alpha vs. beta approached 5, which is predicted by a 1:1 ratio of alpha to beta. The sorbitol gradient resolved two major membrane samples: a mixture of endoplasmic reticulum and Golgi populations and a plasma membrane-enriched sample. Immature beta (beta i) could not be detected in the plasma membrane-enriched samples at levels greater than could be attributed to cross-contamination by intracellular membranes. Mature beta (beta m) became detectable after 30 min, and conversion of beta i to beta m was 90% complete at 120 min. A peak of labeled alpha-subunit appeared in the plasma membrane-enriched sample at 60 min, coincident with the appearance of labeled beta m-subunit in this sample, suggesting movement as alpha beta-heterodimers.

scholarly journals Glucose represses transcription of Saccharomyces cerevisiae nuclear genes that encode mitochondrial components.

1984 ◽  
Vol 4 (5) ◽  
pp. 939-946 ◽  
Author(s):  
E Szekely ◽  
D L Montgomery
Keyword(s):  
Blot Hybridization ◽  
Beta Subunit ◽  
Alpha Subunit ◽  
Northern Blot Hybridization ◽  
Rich Medium ◽  
Genes Encoding ◽  
Atpase Subunits ◽  
Steady State Levels ◽  
Alpha Beta ◽  
Kinetics Of

By Northern blot hybridization analysis, we demonstrated that the steady-state levels of mRNAs specifying the alpha subunit of ATPase, the beta subunit of ATPase, and the ATP/ADP translocator are all reduced in cells grown in glucose-rich medium. The extent to which glucose represses the levels of alpha, beta, and translocator mRNAs varies from strain to strain, from 2.5- to 7-fold. Furthermore, by hybridization experiments with an excess of DNA, we showed that glucose represses the rates of synthesis of these mRNAs. The kinetics of repression and depression of transcription were also studied. Finally, a mutant was characterized which appears to be defective in depression of transcription of the genes encoding the alpha and beta ATPase subunits as well as the ATP/ADP translocator.

scholarly journals 5-Fluoroindole resistance identifies tryptophan synthase beta subunit mutants in Arabidopsis thaliana.

Genetics ◽  
1995 ◽  
Vol 140 (1) ◽  
pp. 303-313
Author(s):  
A J Barczak ◽  
J Zhao ◽  
K D Pruitt ◽  
R L Last
Keyword(s):  
Arabidopsis Thaliana ◽  
Protein A ◽  
Beta Subunit ◽  
Alpha Subunit ◽  
Anthranilate Synthase ◽  
Tryptophan Synthase ◽  
Wild Type ◽  
Recessive Mutations ◽  
The Stability

Abstract A study of the biochemical genetics of the Arabidopsis thaliana tryptophan synthase beta subunit was initiated by characterization of mutants resistant to the inhibitor 5-fluoroindole. Thirteen recessive mutations were recovered that are allelic to trp2-1, a mutation in the more highly expressed of duplicate tryptophan synthase beta subunit genes (TSB1). Ten of these mutations (trp2-2 through trp2-11) cause a tryptophan requirement (auxotrophs), whereas three (trp2-100 through trp2-102) remain tryptophan prototrophs. The mutations cause a variety of changes in tryptophan synthase beta expression. For example, two mutations (trp2-5 and trp2-8) cause dramatically reduced accumulation of TSB mRNA and immunologically detectable protein, whereas trp2-10 is associated with increased mRNA and protein. A correlation exists between the quantity of mutant beta and wild-type alpha subunit levels in the trp2 mutant plants, suggesting that the synthesis of these proteins is coordinated or that the quantity or structure of the beta subunit influences the stability of the alpha protein. The level of immunologically detectable anthranilate synthase alpha subunit protein is increased in the trp2 mutants, suggesting the possibility of regulation of anthranilate synthase levels in response to tryptophan limitation.

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