The influence of the extracellular domains on the dynamic behavior of membrane proteins

The dynamic behavior of the plasma membrane proteins mediates various cellular processes, such as cell-cell interactions, transmembrane transport and signaling. It is widely accepted that the dynamics of the membrane proteins is determined either by the interactions of the transmembrane domain with the surrounding lipids or by the interaction of the intracellular domain with cytosolic components such as cortical actin. However, the impact of the extracellular domains (ECDs) on the dynamics of membrane proteins is rather unexplored. Here, we investigate how the ECD size influences protein dynamics in lipid bilayer. We reconstitute ECDs of different molecular weights and heights in model membrane systems and analyze ECD-driven protein sorting in lipid domains as well as protein mobility. We observe that increasing the ECD size leads to a decrease in ordered domain partitioning as well as diffusivity. Our data suggests a critical role of the ECDs on membrane protein behavior in the plasma membrane and paves the way to a more complete understanding of membrane protein dynamics that includes interaction with the extracellular matrix and glycocalyx in health and disease.

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Small-residue packing motifs modulate the structure and function of a minimal de novo membrane protein

Scientific Reports ◽

10.1038/s41598-020-71585-8 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Paul Curnow ◽

Benjamin J. Hardy ◽

Virginie Dufour ◽

Christopher J. Arthur ◽

Richard Stenner ◽

...

Keyword(s):

Membrane Proteins ◽

Membrane Protein ◽

Transmembrane Domain ◽

De Novo ◽

Zinc Protoporphyrin ◽

Sequence Motifs ◽

Conserved Sequence ◽

Starting Point ◽

Helix Packing ◽

The Impact

Abstract Alpha-helical integral membrane proteins contain conserved sequence motifs that are known to be important in helix packing. These motifs are a promising starting point for the construction of artificial proteins, but their potential has not yet been fully explored. Here, we study the impact of introducing a common natural helix packing motif to the transmembrane domain of a genetically-encoded and structurally dynamic de novo membrane protein. The resulting construct is an artificial four-helix bundle with lipophilic regions that are defined only by the amino acids L, G, S, A and W. This minimal proto-protein could be recombinantly expressed by diverse prokaryotic and eukaryotic hosts and was found to co-sediment with cellular membranes. The protein could be extracted and purified in surfactant micelles and was monodisperse and stable in vitro, with sufficient structural definition to support the rapid binding of a heme cofactor. The reduction in conformational diversity imposed by this design also enhances the nascent peroxidase activity of the protein-heme complex. Unexpectedly, strains of Escherichia coli expressing this artificial protein specifically accumulated zinc protoporphyrin IX, a rare cofactor that is not used by natural metalloenzymes. Our results demonstrate that simple sequence motifs can rigidify elementary membrane proteins, and that orthogonal artificial membrane proteins can influence the cofactor repertoire of a living cell. These findings have implications for rational protein design and synthetic biology.

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The transcytotic pathway of an apical plasma membrane protein (B10) in hepatocytes is similar to that of IgA and occurs via a tubular pericentriolar compartment

Journal of Cell Science ◽

10.1242/jcs.109.6.1215 ◽

1996 ◽

Vol 109 (6) ◽

pp. 1215-1227 ◽

Cited By ~ 2

Author(s):

I. Hemery ◽

A.M. Durand-Schneider ◽

G. Feldmann ◽

J.P. Vaerman ◽

M. Maurice

Keyword(s):

Electron Microscopy ◽

Plasma Membrane ◽

Membrane Proteins ◽

Membrane Protein ◽

Apical Membrane ◽

Rat Hepatocytes ◽

Apical Plasma Membrane ◽

Apical Surface ◽

Plasma Membrane Protein ◽

Microtubule Disruption

In hepatocytes, newly synthesized apical plasma membrane proteins are first delivered to the basolateral surface and are supposed to reach the apical surface by transcytosis. The transcytotic pathway of apical membrane proteins and its relationship with other endosomal pathways has not been demonstrated morphologically. We compared the intracellular route of an apical plasma membrane protein, B10, with that of polymeric IgA (pIgA), which is transcytosed, transferrin (Tf) which is recycled, and asialoorosomucoid (ASOR) which is delivered to lysosomes. Ligands and anti-B10 monoclonal IgG were linked to fluorochromes or with peroxidase. The fate of each ligand was followed by confocal and electron microscopy in polarized primary monolayers of rat hepatocytes. When fluorescent anti-B10 IgG and fluorescent pIgA were simultaneously endocytosed for 15–30 minutes, they both uniformly labelled a juxtanuclear compartment. By 30–60 minutes, they reached the bile canaliculi. Tf and ASOR were also routed to the juxtanuclear area, but their fluorescence patterns were more punctate. Microtubule disruption prevented all ligands from reaching the juxtanuclear area. This area corresponded, at least partially, to the localization of the mannose 6-phosphate receptor, an endosomal marker. By electron microscopy, the juxtanuclear compartment was made up of anastomosing tubules connected to vacuoles, and was organized around the centrioles. B10 and pIgA were mainly found in the tubules, whereas ASOR was segregated inside the vacuolar elements and Tf within thinner, recycling tubules. In conclusion, transcytosis of the apical membrane protein B10 occurs inside tubules similar to those carrying pIgA, and involves passage via the pericentriolar area. In the pericentriolar area, the transcytotic tubules appear to maintain connections with other endosomal elements where sorting between recycled and degraded ligands occurs.

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

10.1101/2020.08.10.244251 ◽

2020 ◽

Cited By ~ 1

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.

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A Survey of Membrane Proteins in Human Serum

Proteomics Insights ◽

10.4137/pri.s9374 ◽

2012 ◽

Vol 5 ◽

pp. PRI.S9374 ◽

Cited By ~ 2

Author(s):

Nguyen Tien Dung ◽

Phan Van Chi

Keyword(s):

Membrane Proteins ◽

Membrane Protein ◽

Human Serum ◽

Transmembrane Domain ◽

Tissue Sample ◽

Ionization Mass ◽

Post Translational Modifications ◽

Molecular Weights ◽

Normal Human ◽

Nano Liquid Chromatography

Serum and membrane proteins are two of the most attractive targets for proteomic analysis. Previous membrane protein studies tend to focus on tissue sample, while membrane protein studies in serum are still limited. In this study, an analysis of membrane proteins in normal human serum was carried out. Nano-liquid chromatography-electrospray ionization mass spectrometry (NanoLC-ESI-MS/MS) and bioinformatics tools were used to identify membrane proteins. Two hundred and seventeen membrane proteins were detected in the human serum, of which 129 membrane proteins have at least one transmembrane domain (TMD). Further characterizations of identified membrane proteins including their subcellular distributions, molecular weights, post translational modifications, transmembrane domains and average of hydrophobicity, were also implemented. Our results showed the potential of membrane proteins in serum for diagnosis and treatment of diseases.

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Plasma Membrane Protein Nce102 Modulates Morphology and Function of the Yeast Vacuole

Biomolecules ◽

10.3390/biom10111476 ◽

2020 ◽

Vol 10 (11) ◽

pp. 1476

Author(s):

Katarina Vaskovicova ◽

Petra Vesela ◽

Jakub Zahumensky ◽

Dagmar Folkova ◽

Maria Balazova ◽

...

Keyword(s):

Plasma Membrane ◽

Membrane Proteins ◽

Membrane Protein ◽

Vacuolar Membrane ◽

Yeast Culture ◽

Membrane Domains ◽

Biological Functions ◽

Plasma Membrane Protein ◽

Cell Architecture ◽

And Function

Membrane proteins are targeted not only to specific membranes in the cell architecture, but also to distinct lateral microdomains within individual membranes to properly execute their biological functions. Yeast tetraspan protein Nce102 has been shown to migrate between such microdomains within the plasma membrane in response to an acute drop in sphingolipid levels. Combining microscopy and biochemistry methods, we show that upon gradual ageing of a yeast culture, when sphingolipid demand increases, Nce102 migrates from the plasma membrane to the vacuole. Instead of being targeted for degradation it localizes to V-ATPase-poor, i.e., ergosterol-enriched, domains of the vacuolar membrane, analogous to its plasma membrane localization. We discovered that, together with its homologue Fhn1, Nce102 modulates vacuolar morphology, dynamics, and physiology. Specifically, the fusing of vacuoles, accompanying a switch of fermenting yeast culture to respiration, is retarded in the strain missing both proteins. Furthermore, the absence of either causes an enlargement of ergosterol-rich vacuolar membrane domains, while the vacuoles themselves become smaller. Our results clearly show decreased stability of the V-ATPase in the absence of either Nce102 or Fhn1, a possible result of the disruption of normal microdomain morphology of the vacuolar membrane. Therefore, the functionality of the vacuole as a whole might be compromised in these cells.

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A Cold-Sensitive Listeria monocytogenes Mutant Has a Transposon Insertion in a Gene Encoding a Putative Membrane Protein and Shows Altered (p)ppGpp Levels

Applied and Environmental Microbiology ◽

10.1128/aem.02607-05 ◽

2006 ◽

Vol 72 (6) ◽

pp. 3955-3959 ◽

Cited By ~ 37

Author(s):

Siqing Liu ◽

Darrell O. Bayles ◽

Tricia M. Mason ◽

Brian J. Wilkinson

Keyword(s):

Listeria Monocytogenes ◽

Low Temperature ◽

Membrane Protein ◽

Transmembrane Domain ◽

Critical Role ◽

Peptide Sequence ◽

Temperature Adaptation ◽

Signal Peptide Sequence ◽

C Terminus ◽

Cold Sensitive

ABSTRACT A cold-sensitive Listeria monocytogenes mutant designated cld-14 was obtained by transposon Tn917 mutagenesis. The gene interrupted by Tn917 in cld-14 was the L. monocytogenes LMOf2365_1485 homolog, which exhibits 45.7% homology to the Bacillus subtilis yqfF locus. LMOf2365_1485, here designated pgpH, encodes a putative integral membrane protein with a predicted molecular mass of 81 kDa. PgpH is predicted to contain a conserved N-terminal signal peptide sequence, seven transmembrane helices, and a hydrophilic C terminus, which likely extends into the cytosol. The Tn917 insertion in pgpH is predicted to result in production of a premature polypeptide truncated at the fifth transmembrane domain. The C terminus of PgpH, which is probably absent in cld-14, contains a highly conserved HD domain that belongs to a metal-dependent phosphohydrolase family. Strain cld-14 accumulated higher levels of (p)ppGpp than the wild type accumulated, indicating that the function of PgpH may be to adjust cellular (p)ppGpp levels during low-temperature growth. The cld-14pgpH + complemented strain was able to grow at a low temperature, like the parent strain, providing direct evidence that the activity of PgpH is important in low-temperature adaptation. Because of its predicted membrane location, PgpH may play a critical role in sensing the environmental temperature and altering cellular (p)ppGpp levels to allow the organism to adapt to low temperatures.

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Density of newly synthesized plasma membrane proteins in intracellular membranes II. Biochemical studies.

The Journal of Cell Biology ◽

10.1083/jcb.98.6.2142 ◽

1984 ◽

Vol 98 (6) ◽

pp. 2142-2147 ◽

Cited By ~ 109

Author(s):

P Quinn ◽

G Griffiths ◽

G Warren

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Membrane Proteins ◽

Membrane Protein ◽

Semliki Forest Virus ◽

Companion Paper ◽

Intracellular Membranes ◽

Surface Areas ◽

Quantitative Immunoblotting ◽

Total Membrane

Using two independent methods, incorporation of radioactive amino-acid and quantitative immunoblotting, we have determined that the rate of synthesis of each of the Semliki Forest virus (SFV) proteins in infected baby hamster kidney (BHK) cells is 1.2 X 10(5) copies/cell/min. Given the absolute surface areas of the endoplasmic reticulum and Golgi complex presented in the companion paper (Griffiths, G., G. Warren, P. Quinn , O. Mathieu - Costello , and A. Hoppeler , 1984, J. Cell Biol. 98:2133-2141), and the approximate time spent in these organelles during their passage to the plasma membrane (Green J., G. Griffiths, D. Louvard , P. Quinn , and G. Warren 1981, J. Mol. Biol. 152:663-698), the mean density of each viral protein in these organelles can be calculated to be 90 and 750 molecules/micron 2 membrane, respectively. In contrast, we have determined that the density of total endogenous integral membrane proteins in these organelles is approximately 30,000 molecules/micron 2 so that the spike proteins constitute only 0.28 and 2.3% of total membrane protein in the endoplasmic reticulum and Golgi, respectively. Quantitative immunoblotting was used to give direct estimates of the concentrations of one of the viral membrane protein precursors (E1) in subcellular fractions; these agreed closely with the calculated values. The data are discussed with respect to the sorting of transported proteins from those endogenous to the intracellular membranes.

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Tail-Anchored Inner Membrane Protein ElaB Increases Resistance to Stress While Reducing Persistence in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.00057-17 ◽

2017 ◽

Vol 199 (9) ◽

Cited By ~ 11

Author(s):

Yunxue Guo ◽

Xiaoxiao Liu ◽

Baiyuan Li ◽

Jianyun Yao ◽

Thomas K. Wood ◽

...

Keyword(s):

Oxidative Stress ◽

Escherichia Coli ◽

Membrane Proteins ◽

Membrane Protein ◽

Stationary Phase ◽

Transmembrane Domain ◽

Inner Membrane ◽

Content Type ◽

E Coli ◽

Inner Membrane Protein

ABSTRACT Host-associated bacteria, such as Escherichia coli, often encounter various host-related stresses, such as nutritional deprivation, oxidative stress, and temperature shifts. There is growing interest in searching for small endogenous proteins that mediate stress responses. Here, we characterized the small C-tail-anchored inner membrane protein ElaB in E. coli. ElaB belongs to a class of tail-anchored inner membrane proteins with a C-terminal transmembrane domain but lacking an N-terminal signal sequence for membrane targeting. Proteins from this family have been shown to play vital roles, such as in membrane trafficking and apoptosis, in eukaryotes; however, their role in prokaryotes is largely unexplored. Here, we found that the transcription of elaB is induced in the stationary phase in E. coli and stationary-phase sigma factor RpoS regulates elaB transcription by binding to the promoter of elaB. Moreover, ElaB protects cells against oxidative stress and heat shock stress. However, unlike membrane peptide toxins TisB and GhoT, ElaB does not lead to cell death, and the deletion of elaB greatly increases persister cell formation. Therefore, we demonstrate that disruption of C-tail-anchored inner membrane proteins can reduce stress resistance; it can also lead to deleterious effects, such as increased persistence, in E. coli. IMPORTANCE Escherichia coli synthesizes dozens of poorly understood small membrane proteins containing a predicted transmembrane domain. In this study, we characterized the function of the C-tail-anchored inner membrane protein ElaB in E. coli. ElaB increases resistance to oxidative stress and heat stress, while inactivation of ElaB leads to high persister cell formation. We also demonstrated that the transcription of elaB is under the direct regulation of stationary-phase sigma factor RpoS. Thus, our study reveals that small inner membrane proteins may have important cellular roles during the stress response.

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MRTF transcription and Ezrin-dependent plasma membrane blebbing are required for entotic invasion

The Journal of Cell Biology ◽

10.1083/jcb.201702010 ◽

2017 ◽

Vol 216 (10) ◽

pp. 3087-3095 ◽

Cited By ~ 16

Author(s):

Laura Soto Hinojosa ◽

Manuel Holst ◽

Christian Baarlink ◽

Robert Grosse

Keyword(s):

Plasma Membrane ◽

Cell Invasion ◽

Serum Response Factor ◽

Critical Role ◽

Actin Dynamics ◽

Actin Cortex ◽

Tightly Coupled ◽

Related Transcription Factor ◽

Nuclear Shuttling ◽

The Impact

Entosis is a nonapoptotic form of cell death initiated by actomyosin-dependent homotypic cell-in-cell invasion that can be observed in malignant exudates during tumor progression. We previously demonstrated formin-mediated actin dynamics at the rear of the invading cell as well as nonapoptotic plasma membrane (PM) blebbing in this cellular motile process. Although the contractile actin cortex involved in bleb-driven motility is well characterized, a role for transcriptional regulation in this process has not been studied. Here, we explore the impact of the actin-controlled MRTF–SRF (myocardin-related transcription factor–serum response factor) pathway for sustained PM blebbing and entotic invasion. We find that cortical blebbing is tightly coupled to MRTF nuclear shuttling to promote the SRF transcriptional activity required for entosis. Furthermore, PM blebbing triggered SRF-mediated up-regulation of the metastasis-associated ERM protein Ezrin. Notably, Ezrin is sufficient and important to sustain bleb dynamics for cell-in-cell invasion when SRF is suppressed. Our results highlight the critical role of the actin-regulated MRTF transcriptional pathway for bleb-associated invasive motility, such as during entosis.

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Docosahexaenoic fatty acid-containing phospholipids affect plasma membrane susceptibility to disruption by bacterial toxin-induced macroapertures

10.1101/2020.12.23.424114 ◽

2020 ◽

Author(s):

Meng-Chen Tsai ◽

Lucile Fleuriot ◽

Sébastien Janel ◽

David Gonzalez-Rodriguez ◽

Camille Morel ◽

...

Keyword(s):

Plasma Membrane ◽

Large Scale ◽

Umbilical Vein ◽

Critical Role ◽

Membrane Dynamics ◽

Bacterial Toxin ◽

Rhoa Gtpase ◽

Docosahexaenoic Fatty Acid ◽

Umbilical Vein Endothelial Cells ◽

The Impact

AbstractMetabolic studies and animal knockout models point to the critical role of polyunsaturated docosahexaenoic acid (22:6, DHA)-containing phospholipids (PLs) in physiology. Here, we study the impact of DHA-PLs on the dynamics of transendothelial cell macroapertures (TEMs) tunnels triggered by the RhoA GTPase inhibitory exotoxin C3 from Clostridium botulinum. Through lipidomic analyses, we show that primary human umbilical vein endothelial cells (HUVECs) subjected to DHA-diet undergo a 6-fold DHA-PLs enrichment in plasma membrane at the expense of monounsaturated OA-PLs. In contrast, OA-diet had almost no effect on PLs composition. Consequently, DHA treatment increases the nucleation rate of TEMs by 2-fold that we ascribe to a reduction of cell thickness. We reveal that the global transcellular area of cells remains conserved through a reduction of the width and lifetime of TEMs. Altogether, we reveal a homeostasis between plasma membrane DHA-PLs content and large-scale membrane dynamics.

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