scholarly journals Probing biophysical sequence constraints within the transmembrane domains of rhodopsin by deep mutational scanning

2020 ◽  
Vol 6 (10) ◽  
pp. eaay7505 ◽  
Author(s):  
Wesley D. Penn ◽  
Andrew G. McKee ◽  
Charles P. Kuntz ◽  
Hope Woods ◽  
Veronica Nash ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Transmembrane Domain ◽  
Evolutionary Analysis ◽  
Missense Mutations ◽  
Evolutionary Sequence ◽  
Membrane Expression ◽  
Plasma Membrane Expression ◽  
Tm Domain ◽  
And Function ◽  
Sequence Constraints

Membrane proteins must balance the sequence constraints associated with folding and function against the hydrophobicity required for solvation within the bilayer. We recently found the expression and maturation of rhodopsin are limited by the hydrophobicity of its seventh transmembrane domain (TM7), which contains polar residues that are essential for function. On the basis of these observations, we hypothesized that rhodopsin’s expression should be less tolerant of mutations in TM7 relative to those within hydrophobic TM domains. To test this hypothesis, we used deep mutational scanning to compare the effects of 808 missense mutations on the plasma membrane expression of rhodopsin in HEK293T cells. Our results confirm that a higher proportion of mutations within TM7 (37%) decrease rhodopsin’s plasma membrane expression relative to those within a hydrophobic TM domain (TM2, 25%). These results in conjunction with an evolutionary analysis suggest solvation energetics likely restricts the evolutionary sequence space of polar TM domains.

scholarly journals Loss of P2X7 Receptor Plasma Membrane Expression and Function in Pathogenic B220+ Double-Negative T Lymphocytes of Autoimmune MRL/lpr Mice

PLoS ONE ◽  
2012 ◽  
Vol 7 (12) ◽  
pp. e52161 ◽  
Author(s):  
Sylvain M. Le Gall ◽  
Julie Legrand ◽  
Mohcine Benbijja ◽  
Hanaa Safya ◽  
Karim Benihoud ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
T Lymphocytes ◽  
P2x7 Receptor ◽  
Double Negative ◽  
Membrane Expression ◽  
Plasma Membrane Expression ◽  
And Function

scholarly journals The Trafficking Protein GABARAP Binds to and Enhances Plasma Membrane Expression and Function of the Angiotensin II Type 1 Receptor

2008 ◽  
Vol 102 (12) ◽  
pp. 1539-1547 ◽  
Author(s):  
Julia L. Cook ◽  
Richard N. Re ◽  
Dawn L. deHaro ◽  
Jennifer M. Abadie ◽  
Michelle Peters ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Angiotensin Ii ◽  
Membrane Expression ◽  
Plasma Membrane Expression ◽  
And Function

scholarly journals Mutations in sixth transmembrane domain of AQP2 inhibit its translocation induced by vasopression

2000 ◽  
Vol 278 (3) ◽  
pp. F395-F405 ◽  
Author(s):  
Yumi Yamashita ◽  
Keiji Hirai ◽  
Yoshifumi Katayama ◽  
Kiyohide Fushimi ◽  
Sei Sasaki ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Immunofluorescence Microscopy ◽  
Transmembrane Domain ◽  
Signal Sequence ◽  
Collecting Duct ◽  
Water Channel ◽  
Wild Type ◽  
Membrane Expression ◽  
Plasma Membrane Expression ◽  
Golgi Network

Vasopression-induced phosphorylation of serine 256 of the aquaporin-2 (AQP2) water channel triggers translocation of the protein from cystolic reservoir vesicles to the apical membrane of collecting duct principal cells. Dileucine motifs are located in the sixth transmembrane domain (6TM) of AQP2 and are known as the signal sequence for internalization, sorting from the trans-Golgi network to endosomes/lysosomes, and basolateral sorting. In this study, involvement of 6TM in vasopressin-induced translocation of the protein was investigated. A series of mutations in 6TM of AQP2 was introduced to rat cDNA and expressed in LLC-PK1 cells. Immunofluorescence microscopy indicated that the mutant AQP2 proteins were retained in the cytoplasm after vasopressin stimulation, which actually promoted the plasma membrane expression of wild-type protein. Immunoelectron microscopy showed that the mutant AQP2 proteins reached the endosomes but did not reach the plasma membrane. These results demonstrate that 6TM has essential domains for vasopressin-induced translocation from endosomes to the plasma membrane.

scholarly journals Claudin-19 mediates the effects of NO on the paracellular pathway in thick ascending limbs

2019 ◽  
Vol 317 (2) ◽  
pp. F411-F418
Author(s):  
Casandra M. Monzon ◽  
Jeffrey L. Garvin
Keyword(s):  
Plasma Membrane ◽  
Control Period ◽  
Paracellular Pathway ◽  
Membrane Expression ◽  
No Donor ◽  
Charge Selectivity ◽  
Plasma Membrane Expression ◽  
Cgmp Signaling ◽  
Blocking Antibodies ◽  
Dilution Potential

Claudins are a family of tight junction proteins that provide size and charge selectivity to solutes traversing the paracellular space. Thick ascending limbs (TALs) express numerous claudins, including claudin-19. Nitric oxide (NO), via cGMP, reduces dilution potentials in perfused TALs, a measure of paracellular permeability, but the role of claudin-19 is unknown. We hypothesized that claudin-19 mediates the effects of NO/cGMP on the paracellular pathway in TALs via increases in plasma membrane expression of this protein. We measured the effect of the NO donor spermine NONOate (SPM) on dilution potentials with and without blocking antibodies and plasma membrane expression of claudin-19. During the control period, the dilution potential was −18.2 ± 1.8 mV. After treatment with 200 μmol/l SPM, it was −14.7 ± 2.0 mV ( P < 0.04). In the presence of claudin-19 antibody, the dilution potential was −12.7 ± 2.1 mV. After SPM, it was −12.9 ± 2.4 mV, not significantly different. Claudin-19 antibody alone had no effect on dilution potentials. In the presence of Tamm-Horsfall protein antibody, SPM reduced the dilution potential from −9.7 ± 1.0 to −6.3 ± 1.1 mV ( P < 0.006). Dibutyryl-cGMP (500 µmol/l) reduced the dilution potential from −19.6 ± 2.6 to −17.2 ± 2.3 mV ( P < 0.002). Dibutyryl-cGMP increased expression of claudin-19 in the plasma membrane from 29.9 ± 3.8% to 65.9 ± 10.1% of total ( P < 0.011) but did not change total expression. We conclude that claudin-19 mediates the effects of the NO/cGMP signaling cascade on the paracellular pathway.

scholarly journals Counterregulation of clathrin-mediated endocytosis by the actin and microtubular cytoskeleton in human neutrophils

2009 ◽  
Vol 296 (4) ◽  
pp. C857-C867 ◽  
Author(s):  
Silvia M. Uriarte ◽  
Neelakshi R. Jog ◽  
Gregory C. Luerman ◽  
Samrath Bhimani ◽  
Richard A. Ward ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Actin Cytoskeleton ◽  
Human Neutrophils ◽  
Functional Responses ◽  
Membrane Expression ◽  
Granule Exocytosis ◽  
Plasma Membrane Expression ◽  
Microtubular Cytoskeleton ◽  
Latrunculin A ◽  
Azurophil Granule

We have recently reported that disruption of the actin cytoskeleton enhanced N-formylmethionyl-leucyl-phenylalanine (fMLP)-stimulated granule exocytosis in human neutrophils but decreased plasma membrane expression of complement receptor 1 (CR1), a marker of secretory vesicles. The present study was initiated to determine if reduced CR1 expression was due to fMLP-stimulated endocytosis, to determine the mechanism of this endocytosis, and to examine its impact on neutrophil functional responses. Stimulation of neutrophils with fMLP or ionomycin in the presence of latrunculin A resulted in the uptake of Alexa fluor 488-labeled albumin and transferrin and reduced plasma membrane expression of CR1. These effects were prevented by preincubation of the cells with sucrose, chlorpromazine, or monodansylcadaverine (MDC), inhibitors of clathrin-mediated endocytosis. Sucrose, chlorpromazine, and MDC also significantly inhibited fMLP- and ionomycin-stimulated specific and azurophil granule exocytosis. Disruption of microtubules with nocodazole inhibited endocytosis and azurophil granule exocytosis stimulated by fMLP in the presence of latrunculin A. Pharmacological inhibition of phosphatidylinositol 3-kinase, ERK1/2, and PKC significantly reduced fMLP-stimulated transferrin uptake in the presence of latrunculin A. Blockade of clathrin-mediated endocytosis had no significant effect on fMLP-stimulated phosphorylation of ERK1/2 in neutrophils pretreated with latrunculin A. From these data, we conclude that the actin cytoskeleton functions to limit microtubule-dependent, clathrin-mediated endocytosis in stimulated human neutrophils. The limitation of clathrin-mediated endocytosis by actin regulates the extent of both specific and azurophilic granule exocytosis.

Optimization and Application of a Biotinylation Method for Quantification of Plasma Membrane Expression of Transporters in Cells

2017 ◽  
Vol 19 (5) ◽  
pp. 1377-1386 ◽  
Author(s):  
Vineet Kumar ◽  
Tot Bui Nguyen ◽  
Beáta Tóth ◽  
Viktoria Juhasz ◽  
Jashvant D. Unadkat
Keyword(s):  
Plasma Membrane ◽  
Membrane Expression ◽  
Plasma Membrane Expression ◽  
In Cells

scholarly journals The role of prolines and glycine in the transmembrane domain of LAT

2020 ◽  
Author(s):  
Daniela Glatzová ◽  
Harsha Mavila ◽  
Maria Chiara Saija ◽  
Tomáš Chum ◽  
Lukasz Cwiklik ◽  
...  
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Transmembrane Domain ◽  
Transmembrane Helix ◽  
Helical Structure ◽  
Surface Expression ◽  
Transmembrane Segment ◽  
Proline Residue ◽  
And Function ◽  
The Impact

ABSTRACTLAT is a critical regulator of T cell development and function. It organises signalling events at the plasma membrane. However, the mechanism, which controls LAT localisation at the plasma membrane is not fully understood. Here, we studied the impact of helix-breaking amino acids, two prolines and one glycine, in the transmembrane segment on localisation and function of LAT. Using in silico analysis, confocal and superresolution imaging and flow cytometry we demonstrate that central proline residue destabilises transmembrane helix by inducing a kink. The helical structure and dynamics is further regulated by glycine and another proline residue in the luminal part of LAT transmembrane domain. Replacement of these residues with aliphatic amino acids reduces LAT dependence on palmitoylation for sorting to the plasma membrane. However, surface expression of these mutants is not sufficient to recover function of non-palmitoylated LAT in stimulated T cells. These data indicate that geometry and dynamics of LAT transmembrane segment regulate its localisation and function in immune cells.

scholarly journals Low Plasma Membrane Expression of the Miltefosine Transport Complex Renders Leishmania braziliensis Refractory to the Drug

2009 ◽  
Vol 53 (4) ◽  
pp. 1305-1313 ◽  
Author(s):  
María P. Sánchez-Cañete ◽  
Luís Carvalho ◽  
F. Javier Pérez-Victoria ◽  
Francisco Gamarro ◽  
Santiago Castanys
Keyword(s):  
Plasma Membrane ◽  
Cutaneous Leishmaniasis ◽  
Oral Drug ◽  
Leishmania Braziliensis ◽  
Β Subunit ◽  
Membrane Expression ◽  
Plasma Membrane Expression ◽  
Highly Sensitive ◽  
P Type ◽  
Transport Complex

ABSTRACT Miltefosine (hexadecylphosphocholine, MLF) is the first oral drug with recognized efficacy against both visceral and cutaneous leishmaniasis. However, some clinical studies have suggested that MLF shows significantly less efficiency against the cutaneous leishmaniasis caused by Leishmania braziliensis. In this work, we have determined the cellular and molecular basis for the natural MLF resistance observed in L. braziliensis. Four independent L. braziliensis clinical isolates showed a marked decrease in MLF sensitivity that was due to their inability to internalize the drug. MLF internalization in the highly sensitive L. donovani species requires at least two proteins in the plasma membrane, LdMT, a P-type ATPase involved in phospholipid translocation, and its β subunit, LdRos3. Strikingly, L. braziliensis parasites showed highly reduced levels of this MLF translocation machinery at the plasma membrane, mainly because of the low expression levels of the β subunit, LbRos3. Overexpression of LbRos3 induces increased MLF sensitivity not only in L. braziliensis promastigotes but also in intracellular amastigotes. These results further highlight the importance of the MLF translocation machinery in determining MLF potency and point toward the development of protocols to routinely monitor MLF susceptibility in geographic areas where L. braziliensis might be prevalent.

scholarly journals Reduced Plasma Membrane Expression of Dysferlin Mutants Is Attributed to Accelerated Endocytosis via a Syntaxin-4-associated Pathway

2010 ◽  
Vol 285 (37) ◽  
pp. 28529-28539 ◽  
Author(s):  
Frances J. Evesson ◽  
Rachel A. Peat ◽  
Angela Lek ◽  
Fabienne Brilot ◽  
Harriet P. Lo ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Expression ◽  
Plasma Membrane Expression ◽  
Syntaxin 4
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