Biogenesis of tight junctions: the C-terminal domain of occludin mediates basolateral targeting

1998 ◽  
Vol 111 (4) ◽  
pp. 511-519 ◽  
Author(s):  
K. Matter ◽  
M.S. Balda
Keyword(s):  
Cell Surface ◽  
Tight Junctions ◽  
Intracellular Transport ◽  
Transmembrane Protein ◽  
Basolateral Membrane ◽  
Chimeric Protein ◽  
Cytoplasmic Domain ◽  
Terminal Domain ◽  
Surface Domains ◽  
Basolateral Targeting

Tight junctions form a morphological and physical border between the apical and the basolateral cell surface domains of epithelial cells; hence assembly of tight junctions could occur from both of the two plasma membrane domains. We show here that the C-terminal cytoplasmic domain of occludin, the only known transmembrane protein of tight junctions, was sufficient to mediate basolateral expression of a chimeric protein. Since this chimera was transported directly to the basolateral membrane during biosynthesis, the C-terminal domain of occludin contains a basolateral targeting signal. Additionally, the C-terminal domain of occludin was also able to mediate endocytosis. Thus, the C-terminal cytoplasmic domain appears to govern intracellular transport of occludin. To test whether the basolateral membrane is an obligatory intermediate in transport of occludin to tight junctions, we analyzed the expression of occludin molecules rendered unable to efficiently integrate into tight junctions by the introduction of N-linked glycosylation sites into the two extracellular loops. Indeed, glycosylated occludin accumulated in the basolateral membrane, supporting a model in which the biogenesis of tight junctions occurs from this cell-surface domain.

scholarly journals Role of Cytoplasmic Domain Serines in Intracellular Trafficking of Furin

2004 ◽  
Vol 15 (6) ◽  
pp. 2884-2894 ◽  
Author(s):  
Florencia B. Schapiro ◽  
Thwe Thwe Soe ◽  
William G. Mallet ◽  
Frederick R. Maxfield
Keyword(s):  
Plasma Membrane ◽  
Steady State ◽  
Transmembrane Protein ◽  
Interleukin 2 ◽  
Chimeric Protein ◽  
Cytoplasmic Domain ◽  
Serine Phosphorylation ◽  
Endocytic Recycling ◽  
Late Endosomes

Furin is a transmembrane protein that cycles between the plasma membrane, endosomes, and the trans-Golgi network, maintaining a predominant distribution in the latter. It has been shown previously that Tac-furin, a chimeric protein expressing the extracellular and transmembrane domains of the interleukin-2 receptor α chain (Tac) and the cytoplasmic domain of furin, is delivered from the plasma membrane to the TGN through late endosomes, bypassing the endocytic recycling compartment. Tac-furin also recycles in a loop between the TGN and late endosomes. Localization of furin to the TGN is modulated by a six-amino acid acidic cluster that contains two phosphorylatable serines (SDSEED). We investigated the role of these serines in the trafficking of Tac-furin by using a mutant chimera in which the SDS sequence was replaced by the nonphosphorylatable sequence ADA (Tac-furin/ADA). Although the mutant construct is internalized and delivered to the TGN, both the postendocytic trafficking and the steady-state distribution were found to differ from the wild-type. In contrast with Tac-furin, Tac-furin/ADA does not enter late endosomes after being internalized. Instead, it traffics with transferrin to the endocytic recycling compartment, and from there it is delivered to the TGN. As with Tac-furin, Tac-furin/ADA is sorted from the TGN into late endosomes at steady state, but its retrieval from the late endosomes to the TGN is inhibited. These results suggest that serine phosphorylation plays an important role in at least two steps of Tac-furin trafficking, acting as an active sorting signal that mediates the selective sorting of Tac-furin into late endosomes after internalization, as well as its retrieval from late endosomes back to the TGN.

scholarly journals CRB3 Binds Directly to Par6 and Regulates the Morphogenesis of the Tight Junctions in Mammalian Epithelial Cells

2004 ◽  
Vol 15 (3) ◽  
pp. 1324-1333 ◽  
Author(s):  
Céline Lemmers ◽  
Didier Michel ◽  
Lydie Lane-Guermonprez ◽  
Marie-Hélène Delgrossi ◽  
Emmanuelle Médina ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Tight Junction ◽  
Tight Junctions ◽  
Transmembrane Protein ◽  
Direct Interaction ◽  
Extracellular Domain ◽  
Cytoplasmic Domain ◽  
Epithelial Morphogenesis ◽  
Neurotrophin Receptor ◽  
Tight Junction Formation

Crumbs is an apical transmembrane protein crucial for epithelial morphogenesis in Drosophila melanogaster embryos. A protein with all the characteristics for a Crumbs homologue has been identified from patients suffering from retinitis pigmentosa group 12, but this protein (CRB1) is only expressed in retina and some parts of the brain, both in human and mouse. Here, we describe CRB3, another Crumbs homologue that is preferentially expressed in epithelial tissues and skeletal muscles in human. CRB3 shares the conserved cytoplasmic domain with other Crumbs but exhibits a very short extracellular domain without the EGF- and laminin A-like G repeats present in the other Crumbs. CRB3 is localized to the apical and subapical area of epithelial cells from the mouse and human intestine, suggesting that it could play a role in epithelial morphogenesis. Indeed, expression of CRB3 or of a chimera containing the extracellular domain of the neurotrophin receptor p75NTR and the transmembrane and cytoplasmic domains of CRB3 led to a slower development of functional tight junctions in Madin-Darby canine kidney cells. This phenotype relied on the presence of CRB3 four last amino acids (ERLI) that are involved in a direct interaction with Par6, a regulator of epithelial polarity and tight junction formation. Thus, CRB3, through its cytoplasmic domain and its interactors, plays a role in apical membrane morphogenesis and tight junction regulation.

scholarly journals Role of the Stable Signal Peptide and Cytoplasmic Domain of G2 in Regulating Intracellular Transport of the Junín Virus Envelope Glycoprotein Complex

2006 ◽  
Vol 80 (11) ◽  
pp. 5189-5198 ◽  
Author(s):  
Sudhakar S. Agnihothram ◽  
Joanne York ◽  
Jack H. Nunberg
Keyword(s):  
Cell Surface ◽  
Signal Peptide ◽  
Envelope Glycoprotein ◽  
Intracellular Transport ◽  
Cytoplasmic Domain ◽  
Virus Envelope ◽  
Quality Control Process ◽  
C Complex ◽  
Stable Signal

ABSTRACT Enveloped viruses utilize the membranous compartments of the host cell for the assembly and budding of new virion particles. In this report, we have investigated the biogenesis and trafficking of the envelope glycoprotein (GP-C) of the Junín arenavirus. The mature GP-C complex is unusual in that it retains a stable signal peptide (SSP) as an essential component in association with the typical receptor-binding (G1) and transmembrane fusion (G2) subunits. We demonstrate that, in the absence of SSP, the G1-G2 precursor is restricted to the endoplasmic reticulum (ER). This constraint is relieved by coexpression of SSP in trans, allowing transit of the assembled GP-C complex through the Golgi and to the cell surface, the site of arenavirus budding. Transport of a chimeric CD4 glycoprotein bearing the transmembrane and cytoplasmic domains of G2 is similarly regulated by SSP association. Truncations to the cytoplasmic domain of G2 abrogate SSP association yet now permit transport of the G1-G2 precursor to the cell surface. Thus, the cytoplasmic domain of G2 is an important determinant for both ER localization and its control through SSP binding. Alanine mutations to either of two dibasic amino acid motifs in the G2 cytoplasmic domain can also mobilize the G1-G2 precursor for transit through the Golgi. Taken together, our results suggest that SSP binding masks endogenous ER localization signals in the cytoplasmic domain of G2 to ensure that only the fully assembled, tripartite GP-C complex is transported for virion assembly. This quality control process points to an important role of SSP in the structure and function of the arenavirus envelope glycoprotein.

scholarly journals Variants of vaccinia virus hemagglutinin altered in intracellular transport.

1986 ◽  
Vol 6 (11) ◽  
pp. 3734-3745 ◽  
Author(s):  
H Shida
Keyword(s):  
Golgi Apparatus ◽  
Cell Surface ◽  
Vaccinia Virus ◽  
Intracellular Transport ◽  
Cytoplasmic Domain ◽  
Slow Movement ◽  
Wild Type ◽  
Sequence Analyses ◽  
In The Wild ◽  
Virus Mutant

Two classes of revertants were isolated from a vaccinia virus mutant whose hemagglutinins (HAs) accumulate on nuclear envelopes and rough endoplasmic reticulums. The HAs of one of the revertants had the same phenotype as the wild type, i.e., rapid and efficient movement to the cell surface. The HAs of the second class had biphasic transport: rapid export to the cell surface as in the wild type and slow movement to the medial cisternae of the Golgi apparatus. Biochemical and nucleotide sequence analyses showed that the HAs of all the mutants examined that have defects in transport from the rough endoplasmic reticulum to the Golgi apparatus have altered cytoplasmic domains and that the HAs of the second class of revertants lack the whole cytoplasmic domain, while the HAs of the first class of revertants have a wild-type cytoplasmic domain.

Cell surface transport, oligomerization, and endocytosis of chimeric type II glycoproteins: role of cytoplasmic and anchor domains

1991 ◽  
Vol 11 (5) ◽  
pp. 2675-2685
Author(s):  
A Kundu ◽  
M A Jabbar ◽  
D P Nayak
Keyword(s):  
Influenza Virus ◽  
Cell Surface ◽  
Mammalian Cells ◽  
Intracellular Transport ◽  
Cytoplasmic Domain ◽  
Type Ii ◽  
Human Influenza Virus ◽  
Chimeric Genes ◽  
Signal Anchor

We investigated the role of cytoplasmic and anchor domains of type II glycoproteins in intracellular transport, oligomerization, and endocytosis by expressing the wild-type and chimeric genes in mammalian cells. Chimeric genes were constructed by exchanging the DNA segments that encode the cytoplasmic and anchor domains between the human influenza virus (A/WSN/33) neuraminidase (NA) and transferrin receptor (TR). The chimeric proteins in which domains were exchanged precisely were productively targeted to the cell surface. However, the proteins appeared to assemble differently in the intracellular compartment. For example, while TR existed predominantly as a dimer, NATR delta 90, containing the cytoplasmic and signal-anchor domains of NA and the ectodomain of TR, was present as a tetramer, a dimer, and a monomer. Similarly, the influenza virus NA existed predominantly as a tetramer but TRNA delta 35, in which the cytoplasmic and signal-anchor domains of TR were joined to the ectodomain of NA, existed predominantly as a dimer, suggesting that the cytoplasmic and anchor domains of type II glycoproteins affect the subunit assembly of heterologous ectodomains. In addition, we analyzed the role of the cytoplasmic domain in endocytosis. NA and NATR delta 90 did not undergo endocytosis, whereas both TR and TRNA delta 35 were internalized efficiently, demonstrating that the NH2 cytoplasmic domain of TR was capable of internalizing a heterologous ectodomain (NA) from the cell surface.

scholarly journals Modulation of Env Content in Virions of Simian Immunodeficiency Virus: Correlation with Cell Surface Expression and Virion Infectivity

2004 ◽  
Vol 78 (13) ◽  
pp. 6775-6785 ◽  
Author(s):  
Eloísa Yuste ◽  
Jacqueline D. Reeves ◽  
Robert W. Doms ◽  
Ronald C. Desrosiers
Keyword(s):  
Cell Surface ◽  
Simian Immunodeficiency Virus ◽  
Stop Codon ◽  
Transmembrane Protein ◽  
Fold Increase ◽  
Surface Expression ◽  
Cytoplasmic Domain ◽  
Cell Surface Expression ◽  
Molar Ratio ◽  
Immunodeficiency Virus

ABSTRACT Specific mutations were created in the cytoplasmic domain of the gp41 transmembrane protein of simian immunodeficiency virus strain 239 (SIV239). The resultant strains included a mutant in which Env residue 767 was changed to a stop codon, a double mutant in which positions 738 and 739 were changed to stop codons, another mutant in which a prominent endocytosis motif was changed from YRPV to GRPV by the substitution of tyrosine 721, and a final combination mutant bearing Q738stop, Q739stop, and Y721G mutations. The effects of these mutations on cell surface expression, on Env incorporation into virions, and on viral infectivity were examined. The molar ratio of Gag to gp120 of 54:1 that we report here for SIV239 virions agrees very well with the ratio of 60:1 reported previously by Chertova et al. (E. Chertova, J. W. Bess, Jr., B. J. Crise, R. C. Sowder II, T. M. Schaden, J. M. Hilburn, J. A. Hoxie, R. E. Benveniste, J. D. Lifson, L. E. Henderson, and L. O. Arthur, J. Virol. 76:5315-5325, 2002), although they were determined by very different methodologies. Assuming 1,200 to 2,500 Gag molecules per virion, this corresponds to 7 to 16 Env trimers per SIV239 virion particle. Although all of the mutations increased Env levels in virions, E767stop had the most dramatic effect, increasing the Env content per virion 25- to 50-fold. Increased levels of Env content in virions correlated strictly with higher levels of Env expression on the cell surface. The increased Env content with the E767stop mutation also correlated with an increased infectivity, but the degree of change was not proportional: the 25- to 50-fold increase in Env content only increased infectivity 2- to 3-fold. All of the mutants replicated efficiently in the CEMx174 and Rh221-89 cell lines. Although some of these findings have been reported previously, our findings show that the effects of the cytoplasmic domain of gp41 on the Env content in virions can be dramatic, that the Env content in virions correlates strictly with the levels of cell surface expression, and that the Env content in virions can determine infectivity; furthermore, our results define a particular change with the most dramatic effects.

scholarly journals The Small GTPase Rab13 Regulates Assembly of Functional Tight Junctions in Epithelial Cells

2002 ◽  
Vol 13 (6) ◽  
pp. 1819-1831 ◽  
Author(s):  
Anne-Marie Marzesco ◽  
Irene Dunia ◽  
Rudy Pandjaitan ◽  
Michel Recouvreur ◽  
Daniel Dauzonne ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Cell Surface ◽  
Tight Junctions ◽  
Mdck Cells ◽  
Transmembrane Protein ◽  
Freeze Fracture ◽  
Small Gtpase ◽  
Cell Contact ◽  
Junctional Complexes ◽  
And Function

Junctional complexes such as tight junctions (TJ) and adherens junctions are required for maintaining cell surface asymmetry and polarized transport in epithelial cells. We have shown that Rab13 is recruited to junctional complexes from a cytosolic pool after cell–cell contact formation. In this study, we investigate the role of Rab13 in modulating TJ structure and functions in epithelial MDCK cells. We generate stable MDCK cell lines expressing inactive (T22N mutant) and constitutively active (Q67L mutant) Rab13 as GFP-Rab13 chimeras. Expression of GFP-Rab13Q67L delayed the formation of electrically tight epithelial monolayers as monitored by transepithelial electrical resistance (TER) and induced the leakage of small nonionic tracers from the apical domain. It also disrupted the TJ fence diffusion barrier. Freeze-fracture EM analysis revealed that tight junctional structures did not form a continuous belt but rather a discontinuous series of stranded clusters. Immunofluorescence studies showed that the expression of Rab13Q67L delayed the localization of the TJ transmembrane protein, claudin1, at the cell surface. In contrast, the inactive Rab13T22N mutant did not disrupt TJ functions, TJ strand architecture nor claudin1 localization. Our data revealed that Rab13 plays an important role in regulating both the structure and function of tight junctions.

scholarly journals Neurodegeneration-associated mutant TREM2 proteins abortively cycle between the ER and ER–Golgi intermediate compartment

2017 ◽  
Author(s):  
Daniel W. Sirkis ◽  
Renan E. Aparicio ◽  
Randy Schekman
Keyword(s):  
Cell Surface ◽  
Intracellular Transport ◽  
Transmembrane Protein ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Vesicle Budding ◽  
Control Center ◽  
Mutant Proteins ◽  
Intermediate Compartment ◽  
Er Export

ABSTRACTTriggering receptor expressed on myeloid cells 2 (TREM2) is a transmembrane protein expressed on microglia within the brain. Several rare mutations in TREM2 cause an early-onset form of neurodegeneration when inherited homozygously. Here we investigate how these mutations affect the intracellular transport of TREM2. We find that most pathogenic TREM2 mutant proteins fail to undergo normal maturation in the Golgi complex and show markedly reduced cell surface expression. Prior research has suggested that two such mutants are retained in the endoplasmic reticulum (ER), but we find, using a cell-free COPII vesicle budding reaction, that mutant TREM2 is exported efficiently from the ER. In addition, mutant TREM2 becomes sensitive to cleavage by endoglycosidase D under conditions that inhibit recycling to the ER, indicating that it normally reaches a post-ER compartment. Maturation-defective TREM2 mutants are also efficiently bound by a lectin that recognizes O-glycans added in the ER–Golgi intermediate compartment (ERGIC) and cis Golgi cisterna. Finally, mutant TREM2 accumulates in the ERGIC in cells depleted of COPI. These results indicate that efficient ER export is not sufficient to enable normal cell surface expression of TREM2. Moreover, our findings suggest that the ERGIC may play an underappreciated role as a quality-control center for mutant and/or malformed membrane proteins.

scholarly journals An Endocytosed TGN38 Chimeric Protein Is Delivered to the TGN after Trafficking through the Endocytic Recycling Compartment in CHO Cells

1998 ◽  
Vol 142 (4) ◽  
pp. 923-936 ◽  
Author(s):  
Richik N. Ghosh ◽  
William G. Mallet ◽  
Thwe T. Soe ◽  
Timothy E. McGraw ◽  
Frederick R. Maxfield
Keyword(s):  
Steady State ◽  
Cell Surface ◽  
Cho Cells ◽  
Transmembrane Protein ◽  
Chimeric Protein ◽  
Steady State Distribution ◽  
Early Endosomes ◽  
Simple Kinetic Model ◽  
Microscopy Analysis ◽  
Confocal Microscopy Analysis

To examine TGN38 trafficking from the cell surface to the TGN, CHO cells were stably transfected with a chimeric transmembrane protein, TacTGN38. We used fluorescent and 125I-labeled anti-Tac IgG and Fab fragments to follow TacTGN38's postendocytic trafficking. At steady-state, anti-Tac was mainly in the TGN, but shortly after endocytosis it was predominantly in early endosomes. 11% of cellular TacTGN38 is on the plasma membrane. Kinetic analysis of trafficking of antibodies bound to TacTGN38 showed that after short endocytic pulses, 80% of internalized anti-Tac returned to the cell surface (t1/2 = 9 min), and the remainder trafficked to the TGN. When longer filling pulses and chases were used to load anti-Tac into the TGN, it returned to the cell surface with a t1/2 of 46 min. Quantitative confocal microscopy analysis also showed that fluorescent anti-Tac fills the TGN with a 46-min t1/2. Using the measured rate constants in a simple kinetic model, we predict that 82% of TacTGN38 is in the TGN, and 7% is in endosomes. TacTGN38 leaves the TGN slowly, which accounts for its steady-state distribution despite the inefficient targeting from the cell surface to the TGN.

Sign in / Sign up

Export Citation Format

Share Document