scholarly journals Identification of a cytoplasmic domain important in the polarized expression and clustering of the Kv2.1 K+ channel.

1996 ◽  
Vol 135 (6) ◽  
pp. 1619-1632 ◽  
Author(s):  
R H Scannevin ◽  
H Murakoshi ◽  
K J Rhodes ◽  
J S Trimmer
Keyword(s):  
Amino Acids ◽  
Influenza Virus ◽  
Mdck Cells ◽  
Chimeric Protein ◽  
High Density ◽  
K Channel ◽  
Wild Type ◽  
Lateral Membrane ◽  
Channel Proteins ◽  
Hemagglutinin Protein

The voltage-sensitive K+ channel Kv2.1 has a polarized and clustered distribution in neurons. To investigate the basis for this localization, we expressed wild-type Kv2.1 and two COOH-terminal truncation mutants, delta C318 and delta C187, in polarized epithelial MDCK cells. These functional channel proteins had differing subcellular localization, in that while both wild-type Kv2.1 and delta C187 localized to the lateral membrane in high density clusters, delta C318 was expressed uniformly on both apical and lateral membranes. A chimeric protein containing the hemagglutinin protein from influenza virus and the region of Kv2.1 that differentiates the two truncation mutants (amino acids 536-666) was also expressed in MDCK cells, where it was found in high density clusters similar to those observed for Kv2.1. Polarized expression and clustering of Kv2.1 correlates with detergent solubility, suggesting that interaction with the detergent insoluble cytoskeleton may be necessary for proper localization of this channel.

scholarly journals A Mutation Linked with Bartter's Syndrome Locks Kir 1.1a (Romk1) Channels in a Closed State

1999 ◽  
Vol 114 (5) ◽  
pp. 685-700 ◽  
Author(s):  
Thomas P. Flagg ◽  
Margaret Tate ◽  
Jean Merot ◽  
Paul A. Welling
Keyword(s):  
Amino Acids ◽  
Fluorescent Protein ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
K Channel ◽  
Bartter’S Syndrome ◽  
Wild Type ◽  
Bartter's Syndrome ◽  
Closed State ◽  
Negative Effect

Mutations in the inward rectifying renal K+ channel, Kir 1.1a (ROMK), have been linked with Bartter's syndrome, a familial salt-wasting nephropathy. One disease-causing mutation removes the last 60 amino acids (332–391), implicating a previously unappreciated domain, the extreme COOH terminus, as a necessary functional element. Consistent with this hypothesis, truncated channels (Kir 1.1a 331X) are nonfunctional. In the present study, the roles of this domain were systematically evaluated. When coexpressed with wild-type subunits, Kir 1.1a 331X exerted a negative effect, demonstrating that the mutant channel is synthesized and capable of oligomerization. Plasmalemma localization of Kir 1.1a 331X green fluorescent protein (GFP) fusion construct was indistinguishable from the GFP–wild-type channel, demonstrating that mutant channels are expressed on the oocyte plasma membrane in a nonconductive or locked-closed conformation. Incremental reconstruction of the COOH terminus identified amino acids 332–351 as the critical residues for restoring channel activity and uncovered the nature of the functional defect. Mutant channels that are truncated at the extreme boundary of the required domain (Kir 1.1a 351X) display marked inactivation behavior characterized by frequent occupancy in a long-lived closed state. A critical analysis of the Kir 1.1a 331X dominant negative effect suggests a molecular mechanism underlying the aberrant closed-state stabilization. Coexpression of different doses of mutant with wild-type subunits produced an intermediate dominant negative effect, whereas incorporation of a single mutant into a tetrameric concatemer conferred a complete dominant negative effect. This identifies the extreme COOH terminus as an important subunit interaction domain, controlling the efficiency of oligomerization. Collectively, these observations provide a mechanistic basis for the loss of function in one particular Bartter's-causing mutation and identify a structural element that controls open-state occupancy and determines subunit oligomerization. Based on the overlapping functions of this domain, we speculate that intersubunit interactions within the COOH terminus may regulate the energetics of channel opening.

scholarly journals Endocytosis of chimeric influenza virus hemagglutinin proteins that lack a cytoplasmic recognition feature for coated pits.

1996 ◽  
Vol 134 (2) ◽  
pp. 339-348 ◽  
Author(s):  
J Lazarovits ◽  
H Y Naim ◽  
A C Rodriguez ◽  
R H Wang ◽  
E Fire ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Cell Surface ◽  
Dna Sequences ◽  
Chimeric Protein ◽  
Amino Acid Sequences ◽  
Surface Proteins ◽  
Wild Type ◽  
Coated Pits ◽  
External Domain ◽  
Simplex Virus

The influenza virus A/Japan/305/57 hemagglutinin (HA) can be converted from a protein that is essentially excluded from coated pits into one that is internalized at approximately the rate of uptake of bulk membrane by replacing the HA transmembrane and cytoplasmic sequences with those of either of two other glycoproteins (Roth et al., 1986. J. Cell Biol. 102:1271-1283). To identify more precisely the foreign amino acid sequences responsible for this change in HA traffic, DNA sequences encoding the transmembrane (TM) or cytoplasmic (CD) domains of either the G glycoprotein of vesicular stomatitis virus (VSV) or the gC glycoprotein of herpes simplex virus were exchanged for those encoding the analogous regions of wild type HA (HA wt). HA-HA-G and HA-HA-gC, chimeras that contain only a foreign CD, resembled HA wt in having a long residence on the cell surface and were internalized very slowly. HA-HA-gC was indistinguishable from HA in our assays, whereas twice as much HA-HA-G was internalized as was HA wt. However, HA-G-HA, containing only a foreign TM, was internalized as efficiently as was HA-G-G, a chimeric protein with transmembrane and cytoplasmic sequences of VSV G protein. Conditions that blocked internalization through coated pits also inhibited endocytosis of the chimeric proteins. Although the external domains of the chimeras were less well folded than that of the wild type HA, denaturation of the wild type HA external domain by treatment with low pH did not increase the interaction of HA with coated pits. However, mutation of four amino acids in the TM of HA allowed the protein to be internalized, indicating that the property that allows HA to escape endocytosis resides in its TM. These results indicate that possession of a cytoplasmic recognition feature is not required for the internalization of all cell surface proteins and suggest that multiple mechanisms for internalization exist that operate at distinctly different rates.

Different properties of two isoforms of annexin XIII in MDCK cells

2000 ◽  
Vol 113 (14) ◽  
pp. 2607-2618 ◽  
Author(s):  
S. Lecat ◽  
P. Verkade ◽  
C. Thiele ◽  
K. Fiedler ◽  
K. Simons ◽  
...  
Keyword(s):  
Amino Acids ◽  
Influenza Virus ◽  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Functional Data ◽  
Mdck Cells ◽  
Amino Terminal ◽  
Influenza Virus Hemagglutinin ◽  
Vesicular Stomatitis ◽  
Amino Terminal Domain

Annexins form a family of proteins that are widely expressed and known to bind membranes in the presence of calcium. Two isoforms of the annexin XIII subfamily are expressed in epithelia. We previously reported that annexin XIIIb is apically localized in MDCK cells and that it is involved in raft-mediated delivery of apical proteins. We have now analyzed the properties of annexin XIIIa, which differs from annexin XIIIb by a deletion of 41 amino acids in the amino-terminal domain, and is distributed both apically and basolaterally. Annexin XIIIa binding to membranes is independent of calcium but requires its myristoyl amino-terminal modification, as observed with annexin XIIIb. Our biochemical and functional data show that annexin XIIIa behaves differently in the apical and in the basolateral compartments. Whereas annexin XIIIa apically can associate with rafts independently of calcium, the basolateral pool requires calcium for this. Annexin XIIIa, like annexin XIIIb, stimulates apical transport of influenza virus hemagglutinin but, in contrast, only annexin XIIIa inhibits basolateral transport of vesicular stomatitis virus G protein. Our results suggest that annexin XIIIa and XIIIb have specific roles in epithelial cells, and because of their structural similarities, these isoforms offer interesting tools for unravelling the functions of annexins.

scholarly journals Basic Residues of the Helix Six Domain of Influenza Virus M1 Involved in Nuclear Translocation of M1 Can Be Replaced by PTAP and YPDL Late Assembly Domain Motifs

2003 ◽  
Vol 77 (12) ◽  
pp. 7078-7092 ◽  
Author(s):  
Eric Ka-Wai Hui ◽  
Subrata Barman ◽  
Tae Yong Yang ◽  
Debi P. Nayak
Keyword(s):  
Amino Acids ◽  
Influenza Virus ◽  
Reverse Genetics ◽  
Infectious Virus ◽  
Nuclear Translocation ◽  
Critical Role ◽  
Site Directed Mutagenesis ◽  
Plaque Morphology ◽  
Wild Type ◽  
Wild Type Phenotype

ABSTRACT Influenza type A virus matrix (M1) protein possesses multiple functional motifs in the helix 6 (H6) domain (amino acids 91 to 105), including nuclear localization signal (NLS) (101-RKLKR-105) involved in translocating M1 from the cytoplasm into the nucleus. To determine the role of the NLS motif in the influenza virus life cycle, we mutated these and the neighboring sequences by site-directed mutagenesis, and influenza virus mutants were generated by reverse genetics. Our results show that infectious viruses were rescued by reverse genetics from all single alanine mutations of amino acids in the H6 domain and the neighboring region except in three positions (K104A and R105A within the NLS motif and E106A in loop 6 outside the NLS motif). Among the rescued mutant viruses, R101A and R105K exhibited reduced growth and small-plaque morphology, and all other mutant viruses showed the wild-type phenotype. On the other hand, three single mutations (K104A, K105A, and E106A) and three double mutations (R101A/K102A, K104A/K105A, and K102A/R105A) failed to generate infectious virus. Deletion (ΔYRKL) or mutation (4A) of YRKL also abolished generation of infectious virus. However, replacement of the YRKL motif with PTAP or YPDL as well as insertion of PTAP after 4A mutation yielded infectious viruses with the wild-type phenotype. Furthermore, mutant M1 proteins (R101A/K102A, ΔYRKL, 4A, PTAP, 4A+PTAP, and YPDL) when expressed alone from cloned cDNAs were only cytoplasmic, whereas the wild-type M1 expressed alone was both nuclear and cytoplasmic as expected. These results show that the nuclear translocation function provided by the positively charged residues within the NLS motif does not play a critical role in influenza virus replication. Furthermore, these sequences of H6 domain can be replaced by late (L) domain motifs and therefore may provide a function similar to that of the L domains of other negative-strand RNA and retroviruses.

scholarly journals Genetic Diversity of Potassium Ion Channel Proteins Encoded by Chloroviruses That Infect Chlorella heliozoae

Viruses ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 678
Author(s):  
Carter R. Murry ◽  
Irina V. Agarkova ◽  
Jayadri S. Ghosh ◽  
Fiona C. Fitzgerald ◽  
Roger M. Carlson ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Amino Acids ◽  
Dna Sequences ◽  
Transmembrane Domain ◽  
Potassium Ion ◽  
K Channel ◽  
K Channels ◽  
Large Plaque ◽  
Channel Proteins ◽  
Dsdna Viruses

Chloroviruses are large, plaque-forming, dsDNA viruses that infect chlorella-like green algae that live in a symbiotic relationship with protists. Chloroviruses have genomes from 290 to 370 kb, and they encode as many as 400 proteins. One interesting feature of chloroviruses is that they encode a potassium ion (K+) channel protein named Kcv. The Kcv protein encoded by SAG chlorovirus ATCV-1 is one of the smallest known functional K+ channel proteins consisting of 82 amino acids. The KcvATCV-1 protein has similarities to the family of two transmembrane domain K+ channel proteins; it consists of two transmembrane α-helixes with a pore region in the middle, making it an ideal model for studying K+ channels. To assess their genetic diversity, kcv genes were sequenced from 103 geographically distinct SAG chlorovirus isolates. Of the 103 kcv genes, there were 42 unique DNA sequences that translated into 26 new Kcv channels. The new predicted Kcv proteins differed from KcvATCV-1 by 1 to 55 amino acids. The most conserved region of the Kcv protein was the filter, the turret and the pore helix were fairly well conserved, and the outer and the inner transmembrane domains of the protein were the most variable. Two of the new predicted channels were shown to be functional K+ channels.

scholarly journals N-Linked Glycans and K147 Residue on Hemagglutinin Synergize To Elicit Broadly Reactive H1N1 Influenza Virus Antibodies

2019 ◽  
Vol 94 (6) ◽  
Author(s):  
Ying Huang ◽  
Simon O. Owino ◽  
Corey J. Crevar ◽  
Donald M. Carter ◽  
Ted M. Ross
Keyword(s):  
Amino Acids ◽  
Influenza Virus ◽  
Binding Site ◽  
Receptor Binding ◽  
Hemagglutination Inhibition ◽  
Vaccine Development ◽  
H1n1 Influenza ◽  
Influenza Viruses ◽  
Wild Type ◽  
Receptor Binding Site

ABSTRACT Vaccination is the most effective way to prevent influenza virus infections. However, the diversity of antigenically distinct isolates is a challenge for vaccine development. In order to overcome the antigenic variability and improve the protective efficacy of influenza vaccines, our research group has pioneered the development of computationally optimized broadly reactive antigens (COBRA) for hemagglutinin (HA). Two candidate COBRA HA vaccines, P1 and X6, elicited antibodies with differential patterns of hemagglutination inhibition (HAI) activity against a panel of H1N1 influenza viruses. In order to better understand how these HA antigens elicit broadly reactive immune responses, epitopes in the Cb, Sa, or Sb antigenic sites of seasonal-like and pandemic-like wild-type or COBRA HA antigens were exchanged with homologous regions in the COBRA HA proteins to determine which regions and residues were responsible for the elicited antibody profile. Mice were vaccinated with virus-like particles (VLPs) expressing one of the 12 modified HA antigens (designated V1 to V12), COBRA HA antigens, or wild-type HA antigens. The elicited antisera was assessed for hemagglutination inhibition activity against a panel of historical seasonal-like and pandemic-like H1N1 influenza viruses. Primarily, the pattern of glycosylation sites and residues in the Sa antigenic region, around the receptor binding site (RBS), served as signatures for the elicitation of broadly reactive antibodies by these HA immunogens. Mice were vaccinated with VLPs expressing HA antigens that lacked a glycosylation site at residue 144 and a deleted lysine at position 147 residue were more effective at protecting against morbidity and mortality following infection with pandemic-like and seasonal-like H1N1 influenza viruses. IMPORTANCE There is a great need to develop broadly reactive or universal vaccines against influenza viruses. Advanced, next-generation hemagglutinin (HA) head-based vaccines that elicit protective antibodies against H1N1 influenza viruses have been developed. This study focused on understanding the specific amino acids around the receptor binding site (RBS) that were important in elicitation of these broadly reactive antibodies. Specific glycan sites and amino acids located at the tip of the HA molecule enhanced the elicitation of these broadly reactive antibodies. A better understanding of the HA structures around the RBS will lead to more effective HA immunogens.

scholarly journals Combinatorial Effect of Two Framework Mutations (E119V and I222L) in the Neuraminidase Active Site of H3N2 Influenza Virus on Resistance to Oseltamivir

2011 ◽  
Vol 55 (6) ◽  
pp. 2942-2952 ◽  
Author(s):  
Mathilde Richard ◽  
Olivier Ferraris ◽  
Alexandra Erny ◽  
Mendy Barthélémy ◽  
Aurélien Traversier ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Reverse Genetics ◽  
Mdck Cells ◽  
Resistance Mechanisms ◽  
Mutant Virus ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Oseltamivir Resistance ◽  
High Level

ABSTRACTNeuraminidase (NA) inhibitors (NIs) are the first line of defense against influenza virus. Reverse genetics experiments allow the study of resistance mechanisms by anticipating the impacts of mutations to the virus. To look at the possibility of an increased effect on the resistance phenotype of a combination of framework mutations, known to confer resistance to oseltamivir or zanamivir, with limited effect on virus fitness, we constructed 4 viruses by reverse genetics in the A/Moscow/10/99 H3N2 background containing double mutations in their neuraminidase genes: E119D+I222L, E119V+I222L, D198N+I222L, and H274Y+I222L (N2 numbering). Among the viruses produced, the E119D+I222L mutant virus was not able to grow without bacterial NA complementation and the D198N+I222L mutant and H274Y+I222L mutant were not stable after passages in MDCK cells. The E119V+I222L mutant was stable after five passages in MDCK cells. This E119V-and-I222L combination had a combinatorial effect on oseltamivir resistance. The total NA activity of the E119V+I222L mutant was low (5% compared to that of the wild-type virus). This drop in NA activity resulted from a decreased NA quantity in the virion in comparison to that of the wild-type virus (1.4% of that of the wild type). In MDCK-SIAT1 cells, the E119V+I222L mutant virus did not present a replicative advantage over the wild-type virus, even in the presence of oseltamivir. Double mutations combining two framework mutations in the NA gene still have to be monitored, as they could induce a high level of resistance to NIs, without impairing the NA affinity. Our study allows a better understanding of the diversity of the mechanisms of resistance to NIs.

scholarly journals Truncation Analysis of TatA and TatB Defines the Minimal Functional Units Required for Protein Translocation

2002 ◽  
Vol 184 (21) ◽  
pp. 5871-5879 ◽  
Author(s):  
Philip A. Lee ◽  
Grant Buchanan ◽  
Nicola R. Stanley ◽  
Ben C. Berks ◽  
Tracy Palmer
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Protein Translocation ◽  
Chimeric Protein ◽  
Transport Activity ◽  
Wild Type ◽  
C Terminus ◽  
Essential Components ◽  
Translocation Pathway ◽  
Terminal Domains

ABSTRACT The TatA and TatB proteins are essential components of the twin arginine protein translocation pathway in Escherichia coli. C-terminal truncation analysis of the TatA protein revealed that a plasmid-expressed TatA protein shortened by 40 amino acids is still fully competent to support protein translocation. Similar truncation analysis of TatB indicated that the final 30 residues of TatB are dispensable for function. Further deletion experiments with TatB indicated that removal of even 70 residues from its C terminus still allowed significant transport. These results imply that the transmembrane and amphipathic helical regions of TatA and TatB are critical for their function but that the C-terminal domains are not essential for Tat transport activity. A chimeric protein comprising the N-terminal region of TatA fused to the amphipathic and C-terminal domains of TatB supports a low level of Tat activity in a strain in which the wild-type copy of either tatA or tatB (but not both) is deleted.

scholarly journals Coupling Assembly of the E-Cadherin/β-Catenin Complex to Efficient Endoplasmic Reticulum Exit and Basal-lateral Membrane Targeting of E-Cadherin in Polarized MDCK Cells

1999 ◽  
Vol 144 (4) ◽  
pp. 687-699 ◽  
Author(s):  
Yih-Tai Chen ◽  
Daniel B. Stewart ◽  
W. James Nelson
Keyword(s):  
Plasma Membrane ◽  
Targeted Delivery ◽  
Mdck Cells ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Chimeric Protein ◽  
Cytoplasmic Domain ◽  
Lateral Membrane ◽  
Sorting Signals ◽  
E Cadherin

The E-cadherin/catenin complex regulates Ca++-dependent cell–cell adhesion and is localized to the basal-lateral membrane of polarized epithelial cells. Little is known about mechanisms of complex assembly or intracellular trafficking, or how these processes might ultimately regulate adhesion functions of the complex at the cell surface. The cytoplasmic domain of E-cadherin contains two putative basal-lateral sorting motifs, which are homologous to sorting signals in the low density lipoprotein receptor, but an alanine scan across tyrosine residues in these motifs did not affect the fidelity of newly synthesized E-cadherin delivery to the basal-lateral membrane of MDCK cells. Nevertheless, sorting signals are located in the cytoplasmic domain since a chimeric protein (GP2CAD1), comprising the extracellular domain of GP2 (an apical membrane protein) and the transmembrane and cytoplasmic domains of E-cadherin, was efficiently and specifically delivered to the basal-lateral membrane. Systematic deletion and recombination of specific regions of the cytoplasmic domain of GP2CAD1 resulted in delivery of <10% of these newly synthesized proteins to both apical and basal-lateral membrane domains. Significantly, >90% of each mutant protein was retained in the ER. None of these mutants formed a strong interaction with β-catenin, which normally occurs shortly after E-cadherin synthesis. In addition, a simple deletion mutation of E-cadherin that lacks β-catenin binding is also localized intracellularly. Thus, β-catenin binding to the whole cytoplasmic domain of E-cadherin correlates with efficient and targeted delivery of E-cadherin to the lateral plasma membrane. In this capacity, we suggest that β-catenin acts as a chauffeur, to facilitate transport of E-cadherin out of the ER and the plasma membrane.

scholarly journals Analysis of transport and targeting of syndecan-1: effect of cytoplasmic tail deletions.

1994 ◽  
Vol 5 (12) ◽  
pp. 1325-1339 ◽  
Author(s):  
H M Miettinen ◽  
S N Edwards ◽  
M Jalkanen
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Cho Cells ◽  
Mdck Cells ◽  
Apical Cell ◽  
Brefeldin A ◽  
Cytoplasmic Tail ◽  
Apparent Molecular Weight ◽  
Wild Type ◽  
Core Proteins

Madin-Darby canine kidney (MDCK) cells and Chinese hamster ovary (CHO) cells were transfected with wild-type and cytoplasmic deletion mutants of mouse syndecan-1 to study the requirements for transport and polarized expression of this proteoglycan. Expression in MDCK cells revealed that wild-type syndecan-1 is directed to the basolateral surface via a brefeldin A-insensitive route. A deletion of the last 12 amino acids of the syndecan-1 cytoplasmic tail (CT22) was sufficient to result in the appearance of mutant proteoglycans at both the basolateral and apical cell surfaces. Treatment with brefeldin A was able to prevent apical transport of the mutants. We thus propose that the C-terminal part of the cytoplasmic tail is required for steady-state basolateral distribution of syndecan-1. In CHO cells a deletion of the last 25 or 33 amino acids of the 34-residue cytoplasmic domain (CT9 and CT1, respectively) resulted in partial retention of the mutants in the endoplasmic reticulum (ER). A deletion mutant lacking the last 12 amino acids (CT22) was not retained. Interestingly, the unglycosylated core proteins of the CT9 and CT1 mutants showed a significantly lower apparent molecular weight when analyzed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis than wild-type syndecan-1. However, when CHO transfectants expressing the CT1 mutant were incubated with brefeldin A, causing fusion of the ER and Golgi, CT1 ran with an almost equally high apparent molecular weight as the wild-type molecule. This would suggest that syndecan-1 undergoes extensive posttranslational modifications or forms an SDS-resistant dimer/complex after transit from the ER.

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