An ion-transporting ATPase encodes multiple apical localization signals.

Gottardi, Cara J.; Caplan, Michael J.

doi:10.1083/jcb.121.2.283

Cited by 127 publications

(63 citation statements)

References 56 publications

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“…GFP fluorescence was generally not observed in the basal membrane that was excluded from analyses. A similar method has previously been used to quantitate the apical to basolateral polarity ratios of Na + -K + -ATPase and H + -K + -ATPase subunits (37). In stably transfected MDCK cells, we obtained similar results using domain selective cell-surface biotinylation and semiquantitative confocal microscopy to determine the ratio of apical to basolateral expression of CFTR (Table 1).…”

Section: Methodssupporting

confidence: 65%

“…Thus, we examined the relative distribution of GFP-CFTR fluorescence in the apical and lateral membranes by semiquantitative confocal microscopy. GFP fluorescence was quantitated in randomly acquired confocal micrograph xz vertical sections as described previously, with minor modifications (37). Using NIH Image software (version 1.57; National Institutes of Health, Bethesda, Maryland, USA), a box (∼2-µm wide and encompassing the length of apical or lateral membranes) was drawn over the region to be measured, and pixel counts within the boxed region were determined.…”

Section: Methodsmentioning

confidence: 99%

“…Mature, glycosylated GFP-wt-CFTR band C is approximately 240 kDa. *High molecular weight form of CFTR that has been reported previously (33,37). (d) Selective biotinylation of the apical membrane.…”

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confidence: 99%

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A PDZ-interacting domain in CFTR is an apical membrane polarization signal

Moyer¹,

Denton²,

Karlson³

et al. 1999

J. Clin. Invest.

268

232

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Polarization of the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel, to the apical plasma membrane of epithelial cells is critical for vectorial transport of chloride in a variety of epithelia, including the airway, pancreas, intestine, and kidney. However, the motifs that localize CFTR to the apical membrane are unknown. We report that the last 3 amino acids in the COOH-terminus of CFTR (T-R-L) comprise a PDZ-interacting domain that is required for the polarization of CFTR to the apical plasma membrane in human airway and kidney epithelial cells. In addition, the CFTR mutant, S1455X, which lacks the 26 COOH-terminal amino acids, including the PDZ-interacting domain, is mispolarized to the lateral membrane. We also demonstrate that CFTR binds to ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50), an apical membrane PDZ domain-containing protein. We propose that COOH-terminal deletions of CFTR, which represent about 10% of CFTR mutations, result in defective vectorial chloride transport, partly by altering the polarized distribution of CFTR in epithelial cells. Moreover, our data demonstrate that PDZ-interacting domains and PDZ domain-containing proteins play a key role in the apical polarization of ion channels in epithelial cells.

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Section: Methodssupporting

confidence: 65%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

A PDZ-interacting domain in CFTR is an apical membrane polarization signal

Moyer¹,

Denton²,

Karlson³

et al. 1999

J. Clin. Invest.

268

232

View full text Add to dashboard Cite

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“…Coexpression studies in LLC-PK1 cells demonstrated the pres-ence of apical sorting signals in both the ␣ and ␤ subunits (17) of the H,K-ATPase and a basolateral sorting signal in the ␣ 1 subunit of the Na,K-ATPase (6). Sorting signals in both Na,KATPase and H,K-ATPase catalytic subunits were shown to reside in their N-terminal halves (6,17).…”

mentioning

confidence: 99%

“…Sorting signals in both Na,KATPase and H,K-ATPase catalytic subunits were shown to reside in their N-terminal halves (6,17). An apical sorting signal of the H,K-ATPase ␤ subunit was demonstrated to be encoded in its N-glycosylation sites (18).…”

mentioning

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

Journal of Biological Chemistry

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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.

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