scholarly journals Intrinsic Signals in the Unique Domain Target p56lckto the Plasma Membrane Independently of CD4

1997 ◽  
Vol 137 (5) ◽  
pp. 1029-1040 ◽  
Author(s):  
Marie-José J.E. Bijlmakers ◽  
Misako Isobe-Nakamura ◽  
Lindsay J. Ruddock ◽  
Mark Marsh
Keyword(s):  
Plasma Membrane ◽  
Protein Tyrosine Kinase ◽  
Fluorescent Protein ◽  
Mdck Cells ◽  
Surface Proteins ◽  
Nih3t3 Cells ◽  
Intrinsic Signals ◽  
Nih 3T3 ◽  
Unique Domain ◽  
Palmitoylation Site

In T lymphocytes, the Src-family protein tyrosine kinase p56lck (Lck) is mostly associated with the cytoplasmic face of the plasma membrane. To determine how this distribution is achieved, we analyzed the location of Lck in lymphoid and in transfected nonlymphoid cells by immunofluorescence. We found that in T cells Lck was targeted correctly, independently of the cell surface proteins CD4 and CD8 with which it interacts. Similarly, in transfected NIH-3T3 fibroblasts, Lck was localized at the plasma membrane, indicating that T cell–specific proteins are not required for targeting. Some variation in subcellular distribution was observed when Lck was expressed in HeLa and MDCK cells. In these cells, Lck associated with both the plasma membrane and the Golgi apparatus, while subsequent expression of CD4 resulted in the loss of Golgi-associated staining. Together, these data indicate that Lck contains intrinsic signals for targeting to the plasma membrane. Furthermore, delivery to this site may be achieved via association with exocytic transport vesicles. A mutant Lck molecule in which the palmitoylation site at cysteine 5 was changed to lysine (LC2) localized to the plasma membrane and the Golgi region in NIH3T3 cells. However, the localization of a mutant in which the palmitoylation site at cysteine 3 was changed to serine (LC1) was indistinguishable from wild-type Lck. Chimeras composed of only the unique domain of Lck linked to either c-Src or the green fluorescent protein similarly localized to the plasma membrane of NIH-3T3 cells. Thus, the targeting of Lck appears to be determined primarily by its unique domain and may be influenced by the use of different palmitoylation sites.

Subcellular redistribution of the renal betaine transporter during hypertonic stress

2003 ◽  
Vol 285 (5) ◽  
pp. C1091-C1100 ◽  
Author(s):  
Stephen A. Kempson ◽  
Vaibhave Parikh ◽  
Lixuan Xi ◽  
Shaoyou Chu ◽  
Marshall H. Montrose
Keyword(s):  
Plasma Membrane ◽  
Posttranscriptional Regulation ◽  
Fluorescent Protein ◽  
Mdck Cells ◽  
Live Cells ◽  
Hypertonic Medium ◽  
Hypertonic Stress ◽  
Antibody Staining ◽  
Renal Inner Medulla ◽  
Green Fluorescent

The betaine transporter (BGT1) protects cells in the hypertonic renal inner medulla by mediating uptake and accumulation of the osmolyte betaine. Transcriptional regulation plays an essential role in upregulation of BGT1 transport when renal cells are exposed to hypertonic medium for 24 h. Posttranscriptional regulation of the BGT1 protein is largely unexplored. We have investigated the distribution of BGT1 protein in live cells after transfection with BGT1 tagged with enhanced green fluorescent protein (EGFP). Fusion of EGFP to the NH2 terminus of BGT1 produced a fusion protein (EGFP-BGT) with transport properties identical to normal BGT1, as determined by ion dependence, inhibitor sensitivity, and apparent Km for GABA. Confocal microscopy of EGFP-BGT fluorescence in transfected Madin-Darby canine kidney (MDCK) cells showed that hypertonic stress for 24 h induced a shift in subcellular distribution from cytoplasm to plasma membrane. This was confirmed by colocalization with anti-BGT1 antibody staining. In fibroblasts, transfected EGFP-BGT caused increased transport in response to hypertonic stress. The activation of transport was not accompanied by increased expression of EGFP-BGT, as determined by Western blotting. Membrane insertion of EGFP-BGT protein in MDCK cells began within 2-3 h after onset of hypertonic stress and was blocked by cycloheximide. We conclude that posttranscriptional regulation of BGT1 is essential for adaptation to hypertonic stress and that insertion of BGT1 protein to the plasma membrane may require accessory proteins.

scholarly journals Quantitative proteomics of MDCK cells identify unrecognized roles of clathrin adaptor AP-1 in polarized distribution of surface proteins

2019 ◽  
pp. 201821076 ◽  
Author(s):  
Paulo S. Caceres ◽  
Diego Gravotta ◽  
Patrick J. Zager ◽  
Noah Dephoure ◽  
Enrique Rodriguez-Boulan
Keyword(s):  
Plasma Membrane ◽  
Isotope Labeling ◽  
Mdck Cells ◽  
Current Model ◽  
Large Population ◽  
Adaptor Proteins ◽  
Surface Proteins ◽  
Experimental Conditions ◽  
Large Populations ◽  
Clathrin Adaptor

The current model of polarized plasma membrane protein sorting in epithelial cells has been largely generated on the basis of experiments characterizing the polarized distribution of a relatively small number of overexpressed model proteins under various experimental conditions. Thus, the possibility exists that alternative roles of various types of sorting machinery may have been underestimated or missed. Here, we utilize domain-selective surface biotinylation combined with stable isotope labeling with amino acids in cell culture (SILAC) and mass spectrometry to quantitatively define large populations of apical and basolateral surface proteins in Madin-Darby canine kidney (MDCK) cells. We identified 313 plasma membrane proteins, of which 38% were apical, 51% were basolateral, and 11% were nonpolar. Silencing of clathrin adaptor proteins (AP) AP-1A, AP-1B, or both caused redistribution of basolateral proteins as expected but also, of a large population of apical proteins. Consistent with their previously reported ability to compensate for one another, the strongest loss of polarity was observed when we silenced AP-1A and AP-1B simultaneously. We found stronger evidence of compensation in the apical pathway compared with the basolateral pathway. Surprisingly, we also found subgroups of proteins that were affected after silencing just one adaptor, indicating previously unrecognized independent roles for AP-1A and AP-1B. While AP-1B silencing mainly affected basolateral polarity, AP-1A silencing seemed to cause comparable loss of apical and basolateral polarity. Our results uncover previously overlooked roles of AP-1 in polarized distribution of apical and basolateral proteins and introduce surface proteomics as a method to examine mechanisms of polarization with a depth not possible until now.

scholarly journals Native Polycystin 2 Functions as a Plasma Membrane Ca2+-Permeable Cation Channel in Renal Epithelia

2003 ◽  
Vol 23 (7) ◽  
pp. 2600-2607 ◽  
Author(s):  
Ying Luo ◽  
Peter M. Vassilev ◽  
Xiaogang Li ◽  
Yoshifumi Kawanabe ◽  
Jing Zhou
Keyword(s):  
Plasma Membrane ◽  
Mdck Cells ◽  
Collecting Duct ◽  
Cation Channel ◽  
Surface Proteins ◽  
Renal Epithelia ◽  
Autosomal Dominant Polycystic Kidney ◽  
Membrane Treatment ◽  
Darby Canine Kidney

ABSTRACT Mutations in polycystin 2 (PC2), a Ca2+-permeable cation channel, cause autosomal dominant polycystic kidney disease. Whether PC2 functions in the endoplasmic reticulum (ER) or in the plasma membrane has been controversial. Here we generated and characterized a polyclonal antibody against PC2, determined the subcellular localization of both endogenous and transfected PC2 by immunohistochemistry and biotinylation of cell surface proteins, and assessed PC2 channel properties with electrophysiology. Endogenous PC2 was found in the plasma membrane and the primary cilium of mouse inner medullar collecting duct (IMCD) cells and Madin-Darby canine kidney (MDCK) cells, whereas heterologously expressed PC2 showed a predominant ER localization. Patch-clamping of IMCD cells expressing endogenous or heterologous PC2 confirmed the presence of the channel on the plasma membrane. Treatment with chaperone-like factors facilitated the translocation of the PC2 channel to the plasma membrane from intracellular pools. The unitary conductances, channel kinetics, and other characteristics of both endogenously and heterologously expressed PC2 were similar to those described in our previous study in Xenopus laevis oocytes. These results show that PC2 functions as a plasma membrane channel in renal epithelia and suggest that PC2 contributes to Ca2+ entry and transport of other cations in defined nephron segments in vivo.

Involvement of CA++ and Calmodulin in the Intracellular Migration of Influenza's Hemagglutinin to the Apical Surface of MDCK Cells

1982 ◽  
Vol 40 ◽  
pp. 30-33
Author(s):  
E. Rodriguez-Boulan ◽  
K.T. Paskiet ◽  
E. Bard
Keyword(s):  
Plasma Membrane ◽  
Mdck Cells ◽  
Viral Envelope ◽  
Surface Proteins ◽  
Apical Surface ◽  
Main Determinant ◽  
Epithelial Cell Lines ◽  
Dog Kidney ◽  
Vesicular Stomatitis ◽  
Integral Proteins

The polarized distribution of surface components between apical and basolateral domains of the plasma membrane constitutes the basis of epithelial function. We are currently studying the mechanisms employed by epithelial cells to segregate different sets of integral proteins in two opposite regions of the plasma membrane. For this purpose, we are utilizing a model system which involves the infection of polarized epithelial cell lines, such as the dog kidney cell line MDCK, with enveloped RNA viruses. Influenza virus and two paramyxoviruses bud from the apical surface regions of MDCK cells, vesicular stomatitis virus (VSV), a rhabdovirus, is assembled instead from the basolateral surface. A main determinant of polarized budding appears to be the addressing of viral envelope glycoproteins to the surface domain that the virus utilizes for budding. The mechanisms and intracellular pathways involved in this sorting are probably the same as those utilized by the cell for its own surface proteins.

scholarly journals Trafficking, Assembly, and Function of a Connexin43-Green Fluorescent Protein Chimera in Live Mammalian Cells

1999 ◽  
Vol 10 (6) ◽  
pp. 2033-2050 ◽  
Author(s):  
Karen Jordan ◽  
Joell L. Solan ◽  
Michel Dominguez ◽  
Michael Sia ◽  
Art Hand ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Gap Junctions ◽  
Gap Junction ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Mdck Cells ◽  
Rat Kidney ◽  
Dye Transfer ◽  
Green Fluorescent

To examine the trafficking, assembly, and turnover of connexin43 (Cx43) in living cells, we used an enhanced red-shifted mutant of green fluorescent protein (GFP) to construct a Cx43-GFP chimera. When cDNA encoding Cx43-GFP was transfected into communication-competent normal rat kidney cells, Cx43-negative Madin–Darby canine kidney (MDCK) cells, or communication-deficient Neuro2A or HeLa cells, the fusion protein of predicted length was expressed, transported, and assembled into gap junctions that exhibited the classical pentalaminar profile. Dye transfer studies showed that Cx43-GFP formed functional gap junction channels when transfected into otherwise communication-deficient HeLa or Neuro2A cells. Live imaging of Cx43-GFP in MDCK cells revealed that many gap junction plaques remained relatively immobile, whereas others coalesced laterally within the plasma membrane. Time-lapse imaging of live MDCK cells also revealed that Cx43-GFP was transported via highly mobile transport intermediates that could be divided into two size classes of <0.5 μm and 0.5–1.5 μm. In some cases, the larger intracellular Cx43-GFP transport intermediates were observed to form from the internalization of gap junctions, whereas the smaller transport intermediates may represent other routes of trafficking to or from the plasma membrane. The localization of Cx43-GFP in two transport compartments suggests that the dynamic formation and turnover of connexins may involve at least two distinct pathways.

Conserved residues F316 and G476 in the concentrative nucleoside transporter 1 (hCNT1) affect guanosine sensitivity and membrane expression, respectively

2005 ◽  
Vol 288 (1) ◽  
pp. C39-C45 ◽  
Author(s):  
Yurong Lai ◽  
Eun-Woo Lee ◽  
Carl C. Ton ◽  
Shashi Vijay ◽  
Huixia Zhang ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Transmembrane Domain ◽  
Mdck Cells ◽  
Site Directed Mutagenesis ◽  
Nucleoside Transporter ◽  
Wild Type ◽  
Membrane Expression ◽  
Concentrative Nucleoside Transporter ◽  
Green Fluorescent

The functional significance of two highly conserved amino acid residues, F316 [putative transmembrane domain (TM)7] and G476 (putative TM11), in the concentrative nucleoside transporter hCNT1 (SLC28A1) was examined by performing site-directed mutagenesis. Conservative mutations at these positions (F316A, F316Y, G476A, and G476L) were generated and expressed in Madin-Darby canine kidney (MDCK) cells as fusion polypeptides with green fluorescent protein (GFP). Unlike wild-type hCNT1, G476A-GFP and G476L-GFP were not expressed in the plasma membrane in undifferentiated or differentiated MDCK cells and had no functional activity. Like wild-type hCNT1, F316A-GFP and F316Y-GFP were expressed in the plasma membrane of undifferentiated MDCK cells and in the apical membrane of differentiated MDCK cells. Remarkably, transport of [3H]uridine by F316Y-GFP or F316A-GFP was highly sensitive to inhibition by guanosine. Furthermore, genotyping of exon 11 of hCNT1 (TM7) in a panel of 260 anonymous human DNA samples revealed a novel F316H variant (TT>CA; 1/260). When expressed in MDCK cells, [3H]uridine transport by F316H was also found to be sensitive to inhibition by guanosine (IC50 = 148 μM). The effect of the F316H mutation resembles the N4 type nucleoside transporter phenotype previously reported to be present in human kidneys. We suggest that the N4 transport system is a naturally occurring variant of hCNT1, perhaps at the F316 position. Collectively, our data show that G476 is important for correct membrane targeting, folding, and/or intracellular processing of hCNT1. In addition, we have discovered that hCNT1 displays natural variation at position F316 and that the variant F316H confers on the transporter an unusual sensitivity to inhibition by guanosine.

scholarly journals The role of aspartic acid residues 405 and 416 of the kidney isotype of sodium-bicarbonate cotransporter 1 in its targeting to the plasma membrane

2012 ◽  
Vol 302 (12) ◽  
pp. C1713-C1730 ◽  
Author(s):  
Hong C. Li ◽  
Volodymyr Kucher ◽  
Emily Y. Li ◽  
Laura Conforti ◽  
Kamyar A. Zahedi ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Sodium Bicarbonate ◽  
Glutamic Acid ◽  
Aspartic Acid ◽  
Functional Activity ◽  
Fluorescent Protein ◽  
Mdck Cells ◽  
Site Directed Mutagenesis ◽  
Sodium Bicarbonate Cotransporter ◽  
Green Fluorescent

The NH2 terminus of the sodium-bicarbonate cotransporter 1 (NBCe1) plays an important role in its targeting to the plasma membrane. To identify the amino acid residues that contribute to the targeting of NBCe1 to the plasma membrane, polarized MDCK cells were transfected with expression constructs coding for green fluorescent protein (GFP)-tagged NBCe1 NH2-terminal deletion mutants, and the localization of GFP-tagged proteins was analyzed by confocal microscopy. Our results indicate that the amino acids between residues 399 and 424 of NBCe1A contain important sequences that contribute to its localization to the plasma membrane. Site-directed mutagenesis studies showed that GFP-NBCe1A mutants D405A and D416A are retained in the cytoplasm of the polarized MDCK epithelial cells. Examination of functional activities of D405A and D416A reveals that their activities are reduced compared with the wild-type NBCe1A. Similarly, aspartic acid residues 449 and 460 of pancreatic NBCe1 (NBCe1B), which correspond to residues 405 and 416 of NBCe1A, are also required for its full functional activity and accurate targeting to the plasma membrane. In addition, while replacement of D416 with glutamic acid did not affect the targeting or functional activity of NBCe1A, substitution of D405 with glutamic acid led to the retention of the mutated protein in the intracellular compartment and impaired functional activity. These studies demonstrate that aspartic acid residues 405 and 416 in the NH2 terminus of NBCe1A are important in its accurate targeting to the plasma membrane.

scholarly journals Myosin Vb Is Associated with Plasma Membrane Recycling Systems

2001 ◽  
Vol 12 (6) ◽  
pp. 1843-1857 ◽  
Author(s):  
Lynne A. Lapierre ◽  
Ravindra Kumar ◽  
Chadwick M. Hales ◽  
Jennifer Navarre ◽  
Sheela G. Bhartur ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Mdck Cells ◽  
Cell Types ◽  
Dominant Negative ◽  
Membrane Recycling ◽  
Recycling System ◽  
Myosin Va ◽  
Myosin Vb ◽  
Recycling Systems

Myosin Va is associated with discrete vesicle populations in a number of cell types, but little is known of the function of myosin Vb. Yeast two-hybrid screening of a rabbit parietal cell cDNA library with dominant active Rab11a (Rab11aS20V) identified myosin Vb as an interacting protein for Rab11a, a marker for plasma membrane recycling systems. The isolated clone, corresponding to the carboxyl terminal 60 kDa of the myosin Vb tail, interacted with all members of the Rab11 family (Rab11a, Rab11b, and Rab25). GFP-myosin Vb and endogenous myosin Vb immunoreactivity codistributed with Rab11a in HeLa and Madin-Darby canine kidney (MDCK) cells. As with Rab11a in MDCK cells, the myosin Vb immunoreactivity was dispersed with nocodazole treatment and relocated to the apical corners of cells with taxol treatment. A green fluorescent protein (GFP)-myosin Vb tail chimera overexpressed in HeLa cells retarded transferrin recycling and caused accumulation of transferrin and the transferrin receptor in pericentrosomal vesicles. Expression of the myosin Vb tail chimera in polarized MDCK cells stably expressing the polymeric IgA receptor caused accumulation of basolaterally endocytosed polymeric IgA and the polymeric IgA receptor in the pericentrosomal region. The myosin Vb tail had no effects on transferrin trafficking in polarized MDCK cells. The GFP-myosin Va tail did not colocalize with Rab11a and had no effects on recycling system vesicle distribution in either HeLa or MDCK cells. The results indicate myosin Vb is associated with the plasma membrane recycling system in nonpolarized cells and the apical recycling system in polarized cells. The dominant negative effects of the myosin Vb tail chimera indicate that this unconventional myosin is required for transit out of plasma membrane recycling systems.

Trafficking of GFP-tagged ΔF508-CFTR to the plasma membrane in a polarized epithelial cell line

2001 ◽  
Vol 281 (6) ◽  
pp. C1889-C1897 ◽  
Author(s):  
Dominique Loffing-Cueni ◽  
Jan Loffing ◽  
Collin Shaw ◽  
Amilyn M. Taplin ◽  
Malu Govindan ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Fluorescent Protein ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Apical Plasma Membrane ◽  
Epithelial Cell Line ◽  
Δf508 Cftr ◽  
Green Fluorescent ◽  
Reduced Temperature

The ΔF508 mutation reduces the amount of cystic fibrosis transmembrane conductance regulator (CFTR) expressed in the plasma membrane of epithelial cells. However, a reduced temperature, butyrate compounds, and “chemical chaperones” allow ΔF508-CFTR to traffic to the plasma membrane and increase Cl− permeability in heterologous and nonpolarized cells. Because trafficking is affected by the polarized state of epithelial cells and is cell-type dependent, our goal was to determine whether these maneuvers induce ΔF508-CFTR trafficking to the apical plasma membrane in polarized epithelial cells. To this end, we generated and characterized a line of polarized Madin-Darby canine kidney (MDCK) cells stably expressing ΔF508-CFTR tagged with green fluorescent protein (GFP). A reduced temperature, glycerol, butyrate, or DMSO had no effect on 8-(4-chlorophenylthio)-cAMP (CPT-cAMP)-stimulated transepithelial Cl− secretion across polarized monolayers. However, when the basolateral membrane was permeabilized, butyrate, but not the other experimental maneuvers, increased the CPT-cAMP-stimulated Cl− current across the apical plasma membrane. Thus butyrate increased the amount of functional ΔF508-CFTR in the apical plasma membrane. Butyrate failed to stimulate transepithelial Cl− secretion because of inhibitory effects on Cl− uptake across the basolateral membrane. These observations suggest that studies on heterologous and nonpolarized cells should be interpreted cautiously. The GFP tag on ΔF508-CFTR will allow investigation of ΔF508-CFTR trafficking in living, polarized MDCK epithelial cells in real time.

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