Endoplasmic Reticulum Quality Control of Oligomeric Membrane Proteins: Topogenic Determinants Involved in the Degradation of the Unassembled Na,K-ATPase α Subunit and in Its Stabilization by β Subunit Assembly

Na؉ /K ؉ -ATPase, a plasma membrane protein abundantly expressed in epithelial tissues, has been identified and linked to numerous biological events, including ion transport and reabsorption. In Na ؉ /K ؉ -ATPase, the ␤-subunit plays a fundamental role in the structural integrity and functional maturation of holoenzyme. Estrogens are important circulating hormones that can regulate Na ؉ /K ؉ -ATPase abundance and activity; however, the specific molecules participating in this process are largely unknown. Here, we characterize that N-myc downstream-regulated gene 2 (NDRG2) is an estrogen up-regulated gene. 17␤-Estradiol binds with estrogen receptor ␤ but not estrogen receptor ␣ to up-regulate NDRG2 expression via transcriptional activation. We also find that NDRG2 interacts with the ␤1-subunit of Na ؉ /K ؉ -ATPase and stabilizes the ␤1-subunit by inhibiting its ubiquitination and degradation. NDRG2-induced prolongation of the ␤1-subunit protein half-life is accompanied by a similar increase in Na ؉ /K ؉ -ATPase-mediated Na ؉ transport and Na ؉ current in epithelial cells. In addition, NDRG2 silencing largely attenuates the accumulation of ␤1-subunit regulated by 17␤-estradiol. Our results demonstrate that estrogen/NDRG2/Na ؉ /K ؉ -ATPase ␤1 pathway is important in promoting Na ؉ /K ؉ -ATPase activity and suggest this novel pathway might have substantial roles in ion transport, fluid balance, and homeostasis. Na ϩ /K ϩ -ATPase, a plasma membrane ion pump, has numerous physiological functions. Of note, it is consisted of three subunits (1), ␣, ␤, and ␥, and the holoenzyme activity required by the participation of the three subunits. ␣ is the catalytic subunit of the enzyme that utilizes ATP hydrolysis to pump K ϩ into the cell in exchange for Na ϩ , which is essential for maintaining normal resting membrane potentials and facilitating the exchange of other materials needed for cellular homeostasis and activity (2-4). ␤-Subunit is responsible for the formation and integrity of the holoenzyme. In vertebrate cells, ␤-subunit may stabilize the correct folding of the ␣-subunit to facilitate its delivery to the plasma membrane (5-7). In addition, evidence showed that ␤-subunit is related to the cell motility and invasion (8, 9). At present, four ␣-isoforms known as ␣1, ␣2, ␣3, ␣4 as well as three different ␤-polypeptides termed as ␤1, ␤2, and ␤3 (3) have been identified. Among these isoforms, ␣1␤1 distributes in nearly every tissue whereas other isoforms exhibit a tissue-specific pattern of expression. ␥-Subunit, a small hydrophobic polypeptide that has only one isoform, is involved in the modulation of Na ϩ /K ϩ -ATPase function (10, 11). The ionic homeostasis maintained by the Na ϩ /K ϩ -ATPase is also critical for cell survival, differentiation, and cell apoptosis (12, 13).As an important molecule in charge of so many biological events, Na ϩ /K ϩ -ATPase is regulated by a number of hormones, including aldosterone, thyroid hormone, glucocorticoid, catecholamines, insulin, carbachol, and androgen. These circulating horm...

Section: Discussionsupporting

confidence: 70%

N-myc Downstream-regulated Gene 2, a Novel Estrogen-targeted Gene, Is Involved in the Regulation of Na+/K+-ATPase

Yang²,

Li³

et al. 2011

“…The Na/K-ATPase ␤ subunit has been generally prescribed to serve two primary roles including 1) a chaperone role that directs the insertion of the ␣ subunit and functional enzyme unit to the appropriate membrane domain (22)(23)(24)(25) and 2) the regulation of cation sensitivity of the Na/K-ATPase (26 -28). Most recently, however, an additional role in regulating epithelial cell phenotype and cell motility has been defined for the Na/K-ATPase ␤ subunit (29 -35).…”

Section: Discussionmentioning

confidence: 99%

Na/K-ATPase β1 Subunit Expression Is Required for Blastocyst Formation and Normal Assembly of Trophectoderm Tight Junction-associated Proteins

Madan

Rose

Watson

2007

Na/K-ATPase plays an important role in mediating blastocyst formation. Despite the expression of multiple Na/K-ATPase ␣ and ␤ isoforms during mouse preimplantation development, only the ␣1 and ␤1 isoforms have been localized to the basolateral membrane regions of the trophectoderm. The aim of the present study was to selectively down-regulate the Na/KATPase ␤1 subunit employing microinjection of mouse 1 cell zygotes with small interfering RNA (siRNA) oligos. Experiments comprised of non-injected controls and two groups microinjected with either Stealth TM Na/K-ATPase ␤1 subunit oligos or nonspecific Stealth TM siRNA as control. Development to the 2-, 4-, 8-, and 16-cell and morula stages did not vary between the three groups. However, only 2.3% of the embryos microinjected with Na/K-ATPase ␤1 subunit siRNA oligos developed to the blastocyst stage as compared with 73% for control-injected and 91% for non-injected controls. Na/K-ATPase ␤1 subunit downregulation was validated by employing reverse transcription-PCR and whole-mount immunofluorescence methods to demonstrate that Na/K-ATPase ␤1 subunit mRNAs and protein were not detectable in ␤1 subunit siRNA-microinjected embryos. Aggregation chimera experiments between ␤1 subunit siRNA-microinjected embryos and controls demonstrated that blockade of blastocyst formation was reversible. The distribution of Na/K-ATPase ␣1 and tight junction-associated proteins occludin and ZO-1 were compared among the three treatment groups. No differences in protein distribution were observed between control groups; however, all three polypeptides displayed an aberrant distribution in Na/K-ATPase ␤1 subunit siRNA-microinjected embryos. Our results demonstrate that the ␤1 subunit of the Na/K-ATPase is required for blastocyst formation and that this subunit is also required to maintain a normal Na/K-ATPase distribution and localization of tight junction-associated polypeptides during preimplantation development.

“…Only the ␣ 1 subunits that assemble with the ␤ 1 subunits exit the ER and reach the plasma membrane. It has been shown in various expression systems that the ␣ 1 subunit alone is not able to exit the ER and is degraded (27). If the expressed H,K-ATPase ␤ subunit assembles with the endogenous Na,K-ATPase ␣ 1 subunit in MDCK cells, the amount of the Na,K-ATPase ␣ 1 subunit present in the plasma membrane must be greater in the cells expressing the H,KATPase ␤ subunit compared with nontransfected cells.…”

Section: Fig 1 Steady State Distribution Of the Wild Type (Wt) Or Mmentioning

confidence: 99%

Use of the H,K-ATPase β Subunit to Identify Multiple Sorting Pathways for Plasma Membrane Delivery in Polarized Cells

Vagin

Turdikulova

Yakubov

et al. 2005

A dynamic equilibrium between multiple sorting pathways maintains polarized distribution of plasma membrane proteins in epithelia. To identify sorting pathways for plasma membrane delivery of the gastric H,K-ATPase ␤ subunit in polarized cells, the protein was expressed as a yellow fluorescent protein N-terminal construct in Madin-Darby canine kidney (MDCK) and LLC-PK1 cells. Confocal microscopy and surface-selective biotinylation showed that 80% of the surface amount of the ␤ subunit was present on the apical membrane in LLC-PK1 cells, but only 40% was present in MDCK cells. Nondenaturing gel electrophoresis of the isolated membranes showed that a significant fraction of the H,K-ATPase ␤ subunits associate with the endogenous Na,K-ATPase ␣ 1 subunits in MDCK but not in LLC-PK cells. Hence, co-sorting of the H,K-ATPase ␤ subunit with the Na,K-ATPase ␣ 1 subunit to the basolateral membrane in MDCK cells may determine the differential distribution of the ␤ subunit in these two cell types. The major fraction of unassociated monomeric H,K-ATPase ␤ subunits is detected in the apical membrane. Quantitative analysis showed that half of the apical pool of the ␤ subunit originates directly from the trans-Golgi network and the other half from transcytosis via the basolateral membrane in MDCK cells. A minor fraction of monomeric ␤ subunits detected in the basolateral membrane represents a transient pool of the protein that undergoes transcytosis to the apical membrane. Hence, the steady state distribution of the H,K-ATPase ␤ subunit in polarized cells depends on the balance between (a) direct sorting from the trans-Golgi network, (b) secondary associative sorting with a partner protein, and (c) transcytosis.