scholarly journals The N-Terminal Basolateral Targeting Signal Unlikely Acts Alone in the Differential Trafficking of Membrane Transporters in MDCK Cells

Biochemistry ◽  
2013 ◽  
Vol 52 (30) ◽  
pp. 5103-5116 ◽  
Author(s):  
Shiu-Ming Kuo ◽  
Li-Yuan Wang ◽  
Siyuan Yu ◽  
Christine E. Campbell ◽  
Sujith A. Valiyaparambil ◽  
...  
Keyword(s):  
Mdck Cells ◽  
Membrane Transporters ◽  
Targeting Signal ◽  
Basolateral Targeting

Intracellular traffic of the ecto-nucleotide pyrophosphatase/phosphodiesterase NPP3 to the apical plasma membrane of MDCK and Caco-2 cells: apical targeting occurs in the absence of N-glycosylation

2000 ◽  
Vol 113 (23) ◽  
pp. 4193-4202 ◽  
Author(s):  
N.R. Meerson ◽  
V. Bello ◽  
J.L. Delaunay ◽  
T.A. Slimane ◽  
D. Delautier ◽  
...  
Keyword(s):  
Cell Surface ◽  
Mdck Cells ◽  
Transmembrane Proteins ◽  
Cell Types ◽  
Apical Plasma Membrane ◽  
Apical Surface ◽  
Intracellular Traffic ◽  
Transmembrane Glycoprotein ◽  
Direct Demonstration ◽  
Targeting Signal

Glycosylation was considered the major signal candidate for apical targeting of transmembrane proteins in polarized epithelial cells. However, direct demonstration of the role of glycosylation has proved difficult because non-glycosylated apical transmembrane proteins usually do not reach the cell surface. Here we were able to follow the targeting of the apical transmembrane glycoprotein NPP3 both when glycosylated and non-glycosylated. Transfected in polarized MDCK and Caco-2 cells, NPP3 was exclusively expressed at the apical membrane. The transport kinetics of the protein to the cell surface were studied after metabolic (35)S-labeling and surface immunoprecipitation. The newly synthesized protein was mainly targeted directly to the apical surface in MDCK cells, whereas 50% transited through the basolateral surface in Caco-2 cells. In both cell types, the basolaterally targeted pool was effectively transcytosed to the apical surface. In the presence of tunicamycin, NPP3 was not N-glycosylated. The non-glycosylated protein was partially retained intracellularly but the fraction that reached the cell surface was nevertheless predominantly targeted apically. However, transcytosis of the non-glycosylated protein was partially impaired in MDCK cells. These results provide direct evidence that glycosylation cannot be considered an apical targeting signal for NPP3, although glycosylation is necessary for correct trafficking of the protein to the cell surface.

scholarly journals Calmodulin Binds to the Basolateral Targeting Signal of the Polymeric Immunoglobulin Receptor

1996 ◽  
Vol 271 (3) ◽  
pp. 1336-1342 ◽  
Author(s):  
Steven J. Chapin ◽  
Carlos Enrich ◽  
Benjamin Aroeti ◽  
Richard J. Havel ◽  
Keith E. Mostov
Keyword(s):  
Polymeric Immunoglobulin Receptor ◽  
Immunoglobulin Receptor ◽  
Targeting Signal ◽  
Basolateral Targeting

scholarly journals Selective inhibition of protein targeting to the apical domain of MDCK cells by brefeldin A.

1992 ◽  
Vol 118 (1) ◽  
pp. 51-62 ◽  
Author(s):  
S H Low ◽  
B L Tang ◽  
S H Wong ◽  
W Hong
Keyword(s):  
Protein Targeting ◽  
Mdck Cells ◽  
Brefeldin A ◽  
Selective Inhibition ◽  
Surface Expression ◽  
Apical Surface ◽  
Golgi Transport ◽  
Apical Domain ◽  
Golgi Network ◽  
Basolateral Targeting

Dipeptidyl peptidase IV (DPPIV) is mainly vectorially targeted to the apical surface in MDCK cells. BFA was found to abolish the apical targeting of DPPIV. This BFA effect could be achieved under conditions where the ER to Golgi transport and the total surface expression of DPPIV were essentially unaffected. BFA executed its effect during the transport from the trans-Golgi network (TGN) to the surface. The inhibition of apical targeting resulted in enhanced mis-targeting to the basolateral surface. The mistargeted DPPIV was transcytosed back to the apical domain only after BFA withdrawal. In contrast, the basolateral targeting of uvomorulin was unaffected by BFA. These results established that the apical targeting of DPPIV was selectively abolished by BFA.

Sorting of MHC class II molecules and the associated invariant chain (Ii) in polarized MDCK cells

1997 ◽  
Vol 110 (5) ◽  
pp. 597-609 ◽  
Author(s):  
A. Simonsen ◽  
E. Stang ◽  
B. Bremnes ◽  
M. Roe ◽  
K. Prydz ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Antigen Presentation ◽  
Mhc Class Ii ◽  
Mdck Cells ◽  
Intracellular Localization ◽  
Cytoplasmic Tail ◽  
Class Ii ◽  
Invariant Chain ◽  
Basolateral Targeting ◽  
Mhc Class Ii Molecules

Epithelial cells have been found to express MHC class II molecules in vivo and are able to perform class II-restricted antigen presentation. The precise intracellular localization of these molecules in epithelial cells has been a matter of debate. We have analyzed the polarized targeting of human MHC class II molecules and the associated invariant chain (Ii) in stably transfected MDCK cells. The class II molecules are located at the basolateral surface and in intracellular vesicles, both when expressed alone or together with Ii. Ii is located in basolateral endosomes and can internalize through the basolateral plasma membrane domain. We show that the cytoplasmic tail of Ii contains information for basolateral targeting as it is sufficient to redirect the apical protein neuraminidase (NA) to the basolateral surface. We find that the two leucine-based motifs (LI and ML) in the cytoplasmic tail of Ii are individually sufficient for endosomal sorting and basolateral targeting of Ii in MDCK cells. In addition, basolateral sorting information is located within the 10 membrane-proximal residues of the Ii cytoplasmic tail. As several different signals mediate basolateral sorting of the class II/Ii complex, a polarized distribution of these molecules may be an essential feature of antigen presentation in epithelial cells.

Lin-7 targets the Kir 2.3 channel on the basolateral membrane via a L27 domain interaction with CASK

2007 ◽  
Vol 293 (6) ◽  
pp. C1733-C1741 ◽  
Author(s):  
Christine Alewine ◽  
Bo-young Kim ◽  
Vandana Hegde ◽  
Paul A. Welling
Keyword(s):  
Mdck Cells ◽  
Basolateral Membrane ◽  
Pdz Domain ◽  
Endocytic Pathway ◽  
Membrane Expression ◽  
Interacting Protein ◽  
Pdz Proteins ◽  
Calcium Calmodulin Dependent ◽  
Anchoring Protein ◽  
Basolateral Targeting

Polarized expression of the Kir 2.3 channel in renal epithelial cells is influenced by the opposing activities of two different PDZ proteins. Mammalian Lin-7 (mLin-7) directly interacts with Kir 2.3 to coordinate basolateral membrane expression, whereas the tax interacting protein 1 (TIP-1), composed of a single PDZ domain, competes for interaction with mLin-7 and drives Kir 2.3 into the endocytic pathway. Here we show that the basolateral targeting function of mLin-7 depends on its L27 domain, which directs interaction with a cognate L27 domain in the basolateral membrane-anchoring protein, calcium/calmodulin-dependent serine protein kinase (CASK). In MDCK cells, the expression of an mLin-7 mutant that lacks the L27 domain displaced Kir 2.3 from the mLin-7/CASK complex and caused the channel to accumulate into large intracellular vesicles that partially colocalized with Rab-11. Conversely, transplantation of the mLin-7 L27 domain to TIP-1 conferred CASK interaction and basolateral targeting of Kir 2.3. Expression of the CASK L27 domain redistributed endogenous mLin-7 to an intracellular compartment and caused Kir 2.3 to accumulate in subapical endosomes. Taken together, these data support a model whereby mLin-7 acts as a PDZ-to-L27 adapter, mediating indirect association of Kir 2.3 with a basolateral membrane scaffold and thereby stabilizing Kir 2.3 at the basolateral membrane.

scholarly journals Glycosylphosphatidylinositol-anchored proteins are preferentially targeted to the basolateral surface in Fischer rat thyroid epithelial cells.

1993 ◽  
Vol 121 (5) ◽  
pp. 1031-1039 ◽  
Author(s):  
C Zurzolo ◽  
M P Lisanti ◽  
I W Caras ◽  
L Nitsch ◽  
E Rodriguez-Boulan
Keyword(s):  
Epithelial Cells ◽  
Cell Lines ◽  
Mdck Cells ◽  
Intestinal Cell ◽  
Gpi Anchor ◽  
Targeting Signal ◽  
Gpi Proteins ◽  
Rat Thyroid ◽  
Striking Contrast ◽  
Intestinal Cell Lines

Glycosylphosphatidylinositol (GPI) acts as an apical targeting signal in MDCK cells and other kidney and intestinal cell lines. In striking contrast with these model polarized cell lines, we show here that Fischer rat thyroid (FRT) epithelial cells do not display a preferential apical distribution of GPI-anchored proteins. Six out of nine detectable endogenous GPI-anchored proteins were localized on the basolateral surface, whereas two others were apical and one was not polarized. Transfection of several model GPI proteins, previously shown to be apically targeted in MDCK cells, also led to unexpected results. While the ectodomain of decay accelerating factor (DAF) was apically secreted, 50% of the native, GPI-anchored form, of this protein was basolateral. Addition of a GPI anchor to the ectodomain of Herpes simplex gD-1, secreted without polarity, led to basolateral localization of the fusion protein, gD1-DAF. Targeting experiments demonstrated that gD1-DAF was delivered vectorially from the Golgi apparatus to the basolateral surface. These results indicate that FRT cells have fundamental differences with MDCK cells with regard to the mechanisms for sorting GPI-anchored proteins: GPI is not an apical signal but, rather, it behaves as a basolateral signal. The "mutant" behavior of FRT cells may provide clues to the nature of the mechanisms that sort GPI-anchored proteins in epithelial cells.

scholarly journals Characterization of a Di-leucine–based Signal in the Cytoplasmic Tail of the Nucleotide-pyrophosphatase NPP1 That Mediates Basolateral Targeting but not Endocytosis

2001 ◽  
Vol 12 (10) ◽  
pp. 3004-3015 ◽  
Author(s):  
Valérie Bello ◽  
James W. Goding ◽  
Vicki Greengrass ◽  
Adnan Sali ◽  
Valentina Dubljevic ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cytoplasmic Tail ◽  
Full Length ◽  
Apical Surface ◽  
Short Sequence ◽  
Conserved Sequence ◽  
Nucleotide Pyrophosphatase ◽  
Targeting Signal ◽  
Basolateral Targeting

Enzymes of the nucleotide pyrophosphatase/phosphodiesterase (NPPase) family are expressed at opposite surfaces in polarized epithelial cells. We investigated the targeting signal of NPP1, which is exclusively expressed at the basolateral surface. Full-length NPP1 and different constructs and mutants were transfected into the polarized MDCK cell line. Expression of the proteins was analyzed by confocal microscopy and surface biotinylation. The basolateral signal of NPP1 was identified as a di-leucine motif located in the cytoplasmic tail. Mutation of either or both leucines largely redirected NPP1 to the apical surface. Furthermore, addition of the conserved sequence AAASLLAP redirected the apical nucleotide pyrophosphatase/phosphodiesterase NPP3 to the basolateral surface. Full-length NPP1 was not significantly internalized. However, when the cytoplasmic tail was deleted upstream the di-leucine motif or when the six upstream flanking amino acids were deleted, the protein was mainly found intracellularly. Endocytosis experiments indicated that these mutants were endocytosed from the basolateral surface. These results identify the basolateral signal of NPP1 as a short sequence including a di-leucine motif that is dominant over apical determinants and point to the importance of surrounding amino acids in determining whether the signal will function as a basolateral signal only or as an endocytotic signal as well.

scholarly journals Cytoplasmic sequence required for basolateral targeting of LDL receptor in livers of transgenic mice

1992 ◽  
Vol 117 (1) ◽  
pp. 39-46 ◽  
Author(s):  
M Yokode ◽  
RK Pathak ◽  
RE Hammer ◽  
MS Brown ◽  
JL Goldstein ◽  
...  
Keyword(s):  
Amino Acid ◽  
Transgenic Mice ◽  
Mdck Cells ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Alpha Helix ◽  
Mutant Receptor ◽  
Amino Acid Stretch ◽  
Basolateral Targeting

When expressed in livers of transgenic mice, the human low density lipoprotein (LDL) receptor is specifically targeted to the basolateral (sinusoidal) surface of hepatocytes as determined by immunofluorescence and immunoelectron microscopy. The COOH-terminal cytoplasmic domain of the receptor (residues 790-839) contains a signal for this targeting. A mutant receptor truncated at residue 812 was localized exclusively to the apical (bile canalicular) surface. A mutant receptor terminating at residue 829 showed the normal basolateral distribution, as did a receptor in which alanine was substituted for serine 833, which was previously shown to be a site for phosphorylation in vitro. These data localize the basolateral targeting signal to the 17-residue segment between residues 812 and 828. A 10-amino acid stretch within this segment shows a 4/10 match with a sequence within a previously identified basolateral sorting motif for the receptor for polymeric IgA/IgM in MDCK cells. The four shared residues are spaced at intervals of three, raising the possibility that they all face the same side of an alpha-helix. We conclude that this 10-amino acid stretch may contain a signal that directs certain proteins, including the LDL receptor and the polymeric IgG/IgM receptor, to the basolateral surface of polarized epithelia.

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