Location of signal sequences for membrane insertion of the Na+,K+-ATPase alpha subunit.

Homareda, Haruo; Kawakami, Kiyoshi; Nagano, Kei

doi:10.1128/mcb.9.12.5742

Cited by 21 publications

(7 citation statements)

References 33 publications

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“…M1 and M3 of the Na,KATPase ␣-subunit exhibit full signal anchor activity, and M2 and M4 exhibit efficient stop transfer function when synthesized in vivo, leading to the formation of membrane-integrated M1/2 and M3/4 membrane pairs. These data are consistent with previous results obtained by in vitro translation of membrane segments of Na,K-ATPase (23,24) as well as of H,KATPase (12). Since each of the four N-terminal segments of H,K-ATPase ␣-subunits can adopt Ncyt or Nout orientations in the membrane (12), it is likely that the native orientation is determined by other factors than sequence information within these topogenic segments.…”

Section: Figsupporting

confidence: 93%

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Membrane Integration of Na,K-ATPase α-Subunits and β-Subunit Assembly

Béguin

Hasler

Beggah

et al. 1998

Journal of Biological Chemistry

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The control of membrane insertion of polytopic proteins is still poorly understood. We carried out in vivo translation/insertion experiments in Xenopus oocytes with combined wild type or mutant membrane segments of the ␣-subunit of the heterodimeric Na,K-ATPase linked to a glycosylation reporter sequence. We confirm that the four N-terminal hydrophobic segments of the ␣-subunit behave as alternating signal anchor/stop transfer motifs necessary for two lipid-inserted membrane pairs. For the six C-terminal membrane segments, however, proper packing depends on specific sequence information and association with the ␤-subunit. M5 is a very inefficient signal anchor sequence due to the presence of prolines and polar amino acids. Its correct membrane insertion is probably mediated by posttranslational hairpin formation with M6, which is favored by a proline pair in the connecting loop. M7 has partial signal anchor function, which may be mediated by the presence of glycine and glutamine residues. The formation of a transmembrane M7/M8 pair requires the association of the ␤-subunit, which induces a conformational change in the connecting extracytoplasmic loop that favors M7/M8 packing. The formation of the M9/M10 pair appears to be predominantly mediated by the efficient stop transfer function of M10. Mutations that provide signal anchor function to M5, M7, and M9 abolish or impede the transport activity of the enzyme. These data illustrate the importance of specific amino acids near or within hydrophobic regions as well as of subunit oligomerization for correct topographical alignment that is necessary for proper folding and/or activity of oligomeric membrane proteins.Many of the important details of the membrane insertion and the maturation of polytopic, oligomeric membrane proteins are still obscure (for reviews, see Refs. 1-3). During the membrane integration, these membrane proteins are subjected to an initial folding process, which is mediated by specific ␣-helical packing (for a review see Ref. 4), interaction with molecular chaperones, and co-translational modifications (for a review, see Ref. 5). Finally, in the case of oligomeric proteins, the maturation process is completed by the assembly of different subunits (for a review, see Ref. 6). Each maturation step is necessary to ultimately result in the correct tertiary structure of oligomeric proteins that is compatible with intracellular transport and function. Analysis of many polytopic membrane proteins is consistent with a model according to which these proteins are produced by the sequential insertion into the endoplasmic reticulum (ER) 1 membrane of signal anchor and stop transfer motifs that generally contain a sequence of about 23 amino acids favoring an ␣-helical conformation and are interrupted by a run of hydrophilic amino acids. The insertion of the first ER-targeting sequence is mediated by the signal recognition particle, which interrupts translation until docked with the signal recognition particle receptor on the ER membrane. Subsequent transmembr...

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Section: Figsupporting

confidence: 93%

“…Cells and Topogenic Effects of ␤-Assembly-As previously reported using in vitro translation (23,24), the four N-terminal membrane segments of the Na,K-ATPase ␣-subunit are efficiently membrane-integrated during synthesis in vivo (Fig. 1).…”

Section: Insertion Competence Of N-and C-terminal Transmembrane Segmementioning

confidence: 60%

Membrane Integration of Na,K-ATPase α-Subunits and β-Subunit Assembly

Béguin

Hasler

Beggah

et al. 1998

Journal of Biological Chemistry

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“…Perhaps post-translational modifications of ATP1A4 are carried out through hydrophobic and electrostatic interactions of membrane-associated molecular chaperones ultimately guiding them to appropriate folding and transportation pathways. Finally, insertion and anchoring of the protein to the plasma membrane could be facilitated by hydrophobic amino acids in the transmembrane domains of ATP1A4 (Homareda et al, 1989). Our study indicates the ability of sperm to synthesize protein suggests the importance of atypical, yet functional translation pathways to meet physiological demands during capacitation and opens interesting areas of investigation in mammalian sperm biology.…”

Section: Capacitation Increases the Content Of Atp1a4 In Bovine Spermmentioning

confidence: 75%

Na/K-ATPase and Regulation of Sperm Function

Thundathil¹,

Rajamanickam²,

Kastelic³

2018

Anim Reprod

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A standard bull breeding soundness evaluation (BBSE) identifies bulls with semen that is grossly abnormal. Nonetheless, semen samples classified as satisfactory based on these traditional approaches differ in fertility; perhaps there are submicroscopic differences in sperm characteristics affecting fertility. Therefore, a better understanding of molecular regulation of sperm function could promote development of novel, evidence-based approaches to predict male fertility. Recently the α4 isoform of Na/K-ATPase (ATP1A4) has received considerable attention, due to its testisspecific expression in post-meiotic germ cells and mature sperm, in addition to its regulation of sperm motility and capacitation. Using fresh bull sperm, we determined that ATP1A4 resided in specialized microdomains (raft and non-raft) of the sperm plasma membrane and activated specific signaling (caveolin-1, EGFR, Src, ERK1/2) molecules during sperm capacitation. Furthermore, ATP1A4 was the predominant isoform responsible for total Na/K-ATPase activity in capacitated sperm. Despite the widely accepted dogma of transcriptional/translational quiescence, bovine sperm translated ATP1A4 mRNA on mitochondrial or mitochondrial-type ribosomes, increasing their content and activity during capacitation. Proteomic analysis of raft and non-raft fractions revealed a significant interaction between ATP1A4 and plakoglobin, a member of the β-catenin family of proteins involved in cell adhesion, in the equatorial segment of capacitated sperm, suggesting a potential role in sperm-oolemma fusion. In frozen-thawed sperm, ATP1A4 content and activity was greater in high-versus low-fertility bulls. Additionally, ATP1A4-induced increases in ROS, calcium, actin polymerization and tyrosine phosphorylation were also involved in regulating post-thaw sperm function in these bulls. Overall, results demonstrated that ATP1A4 had unique roles in controlling several aspects of sperm physiology, acting through well-established enzyme activity and signaling functions. Consequently, isoforms of Na/K-ATPase are potential biomarkers for male fertility.

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“… 12 , 27 ], but as recently pointed out [ 6 ] surprisingly little is still known about the targeting and trafficking of the Na,K-ATPase to the basolateral surface. Both the α and β subunits are assembled at the level of the ER [ 1 , 17 , 18 , 25 ], but if the α subunit is expressed alone it is retained and degraded in the endoplasmic reticulum, while the β subunit is able to traffick alone to the plasma membrane [ 10 , 16 ]. It is likely that the γ subunit becomes synthesized and transported to the basolateral membrane along the same route as the mature α and β subunits, but it has also been expressed alone on the apical surface of mouse blastocysts [ 20 ].…”

Section: Introductionmentioning

confidence: 99%

Expression and Trafficking of the .GAMMA. Subunit of Na,K-ATPase in Hypertonically Challenged IMCD3 Cells

Pihakaski-Maunsbach

Nonaka

Maunsbach

2008

Acta Histochem. Cytochem

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The γ subunit (FXYD2) of Na,K-ATPase is an important regulator of the sodium pump. In this investigation we have analysed the trafficking of γ to the plasma membrane in cultures of inner medullary collecting duct cells (IMCD3) following acute hypertonic challenge and brefeldin A (BFA) treatment. Following hypertonic challenging for 24 hr immunofluorescence labeling revealed initial co-localization of the γ subunit and 58K Golgi protein in the cytoplasm, but no co-localization of α1 and Golgi protein. Exposure of the challenged cells to BFA prevented the subsequent incorporation of γ into the basolateral plasma membrane. The γ subunit instead remained in cytoplasmic vesicles while cell proliferation and cell viability decreased simultaneously. Following removal of BFA from the hypertonic medium the IMCD3 cells recovered with distinct expression of γ in the basolateral membrane. The α1 subunit was only marginally influenced by BFA. The results demonstrate that the γ subunit trafficks to the plasma membrane via the Golgi apparatus, despite the absence of a signal sequence. The results also suggest that the γ and α subunits do not traffic together to the plasma membrane, and that the γ and α subunit have different turnover rates during these experimental conditions.

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Location of signal sequences for membrane insertion of the Na+,K+-ATPase alpha subunit.

Cited by 21 publications

References 33 publications

Membrane Integration of Na,K-ATPase α-Subunits and β-Subunit Assembly

Membrane Integration of Na,K-ATPase α-Subunits and β-Subunit Assembly

Na/K-ATPase and Regulation of Sperm Function

Expression and Trafficking of the .GAMMA. Subunit of Na,K-ATPase in Hypertonically Challenged IMCD3 Cells

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