Weak Dependence of Mobility of Membrane Protein Aggregates on Aggregate Size Supports a Viscous Model of Retardation of Diffusion

Kucik, Dennis F.; Elson, Elliot L.; Sheetz, Michael P.

doi:10.1016/s0006-3495(99)77198-5

Cited by 79 publications

(72 citation statements)

References 40 publications

(82 reference statements)

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“…The aggregation of P450 2C2 observed in the present study, therefore, does not decrease its mobility, as detected by FRAP. These results suggest that parameters other than the state of oligomerization primarily determine lateral mobility in the membranes, consistent with the proposal that there is only a weak correlation between the size and the membrane diffusion coefficient of a protein (36).…”

Section: Discussionsupporting

confidence: 89%

Fluorescence Resonance Energy Transfer Analysis of Cytochromes P450 2C2 and 2E1 Molecular Interactions in Living Cells

Szczesna-Skorupa

Mallah

Kemper

2003

Journal of Biological Chemistry

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The molecular organization of microsomal cytochromes P450 (P450s) and formation of complexes with P450 reductase have been studied previously with isolated proteins and in reconstituted systems. Although these studies demonstrated that some P450s oligomerize in vitro, neither oligomerization nor interactions of P450 with P450 reductase have been studied in living cells. Here we have used fluorescence resonance energy transfer (FRET) to study P450 oligomerization and binding to P450 reductase in live transfected cells. Cytochrome P450 2C2, but not P450 2E1, forms homo-oligomeric structures, and this self-association is mediated by the signal-anchor sequence. Because P450 2C2, in contrast to P450 2E1, is directly retained in the endoplasmic reticulum (ER), these results could suggest that oligomerization may prevent transport from the ER. However, P450 2C1 signal-anchor sequence chimera defective in ER retention also formed oligomers, and chimera containing the cytoplasmic domain of P450 2C2, which is directly retained in the ER, did not exhibit self-oligomerization, which indicates that oligomerization is not correlated with direct retention. By using FRET, we have also detected binding of P450 2C2 and P450 2E1 to P450 reductase. In contrast to self-oligomerization, the catalytic domain can mediate an interaction of P450 2C2 with P450 reductase. These results suggest that microsomal P450s may differ in their quaternary structure but that these differences do not detectably affect interaction with the reductase or transport from the ER.The organization of the cytochrome P450 (P450) 1 -containing monooxygenase system in the microsomal membrane is not well understood despite many studies using a wide variety of biophysical methods. This system consists of P450s, NADPHcytochrome P450 reductase (P450 reductase), and for some P450s, cytochrome b 5 . If P450 levels in the endoplasmic reticulum (ER) are induced to maximal concentrations, there are 20 -30 P450s for each P450 reductase molecule. The presence of multiple forms of P450s in the ER membranes combined with limiting amounts of P450 reductase has important implications for the association of the P450s with each other and the reductase and for electron transfer from the reductase to P450. It has been postulated that either a multimeric complex of P450s binds to a molecule of P450 reductase (1, 2) or that the interaction results from random collisions of independently diffusing monomeric proteins that have high rotational and lateral mobility (3-5). Formation of large complexes by P450s has been observed with both isolated proteins and reconstituted systems (6 -8). Studies on rotational mobilities of P450s in microsomal membranes were also consistent with either self-aggregation or association of P450s with other components of the membranes (9 -11). The second model in which monomeric P450 interacts with P450 reductase is supported by observations that high concentrations of some P450s in membranes result in their aggregation and immobilization, but the addition of...

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Section: Discussionsupporting

confidence: 89%

Fluorescence Resonance Energy Transfer Analysis of Cytochromes P450 2C2 and 2E1 Molecular Interactions in Living Cells

Szczesna-Skorupa

Mallah

Kemper

2003

Journal of Biological Chemistry

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“…The logarithmic function stems from the fact that the diffusion coefficient of membrane proteins often spans several orders of magnitude (Kucik et al, 1999;Thoumine et al, 2005) and uses a reference diffusion coefficient D 0 , the one obtained for Fc-coated beads (0.03 m 2 /s). After checking by immunofluorescence that the amounts of Ncad-Fc adsorbed on microspheres and glass coverslips increased similarly with coating concentration (supplemental Fig.…”

Section: N-cadherin Selectively Stimulates Neurite Extension and Growmentioning

confidence: 99%

A Molecular Clutch between the Actin Flow and N-Cadherin Adhesions Drives Growth Cone Migration

Bard¹,

Boscher²,

Lambert³

et al. 2008

Journal of Neuroscience

137

144

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The adhesion molecule N-cadherin plays important roles in the development of the nervous system, in particular by stimulating axon outgrowth, but the molecular mechanisms underlying this effect are mostly unknown. One possibility, the so-called "molecular clutch" model, could involve a direct mechanical linkage between N-cadherin adhesion at the membrane and intracellular actin-based motility within neuronal growth cones. Using live imaging of primary rat hippocampal neurons plated on N-cadherin-coated substrates and optical trapping of N-cadherin-coated microspheres, we demonstrate here a strong correlation between growth cone velocity and the mechanical coupling between ligand-bound N-cadherin receptors and the retrograde actin flow. This relationship holds by varying ligand density and expressing mutated N-cadherin receptors or small interfering RNAs to perturb binding to catenins. By restraining microsphere motion using optical tweezers or a microneedle, we further show slippage of cadherin-cytoskeleton bonds at low forces, and, at higher forces, local actin accumulation, which strengthens nascent N-cadherin contacts. Together, these data support a direct transmission of actin-based traction forces to N-cadherin adhesions, through catenin partners, driving growth cone advance and neurite extension.

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“…25,26 It was previously believed that the size of a transmembrane object could not be deduced from mobility measurements, as the theoretical work of Saffman and Dellbrück 27 predicted that, within a layer of incompressible fluid, the diffusion coefficient should be insensitive to the size of the diffusing object. Consequently, studies of lateral mobility have neglected model systems 28 where little information should be obtained, and focused on living cells [29][30][31] to explore the mechanical barriers hindering the movement of proteins. The validity of the Saffman-Dellbrück (SD) model has never been adequately verified becauce of a lack of systematic measurements.…”

Section: Introductionmentioning

confidence: 99%

Variation of the Lateral Mobility of Transmembrane Peptides with Hydrophobic Mismatch

et al. 2010

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A hydrophobic mismatch between protein length and membrane thickness can lead to a modification of protein conformation, function, and oligomerization. To study the role of hydrophobic mismatch, we have measured the change in mobility of transmembrane peptides possessing a hydrophobic helix of various length d π in lipid membranes of giant vesicles. We also used a model system where the hydrophobic thickness of the bilayers, h, can be tuned at will. We precisely measured the diffusion coefficient of the embedded peptides and gained access to the apparent size of diffusing objects. For bilayers thinner than d π , the diffusion coefficient decreases, and the derived characteristic sizes are larger than the peptide radii. Previous studies suggest that peptides accommodate by tilting. This scenario was confirmed by ATR-FTIR spectroscopy. As the membrane thickness increases, the value of the diffusion coefficient increases to reach a maximum at h ≈ d π . We show that this variation in diffusion coefficient is consistent with a decrease in peptide tilt. To do so, we have derived a relation between the diffusion coefficient and the tilt angle, and we used this relation to derive the peptide tilt from our diffusion measurements. As the membrane thickness increases, the peptides raise (i.e., their tilt is reduced) and reach an upright position and a maximal mobility for h ≈ d π . Using accessibility measurements, we show that when the membrane becomes too thick, the peptide polar heads sink into the interfacial region. Surprisingly, this "pinching" behavior does not hinder the lateral diffusion of the transmembrane peptides. Ultimately, a break in the peptide transmembrane anchorage is observed and is revealed by a "jump" in the D values.

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Weak Dependence of Mobility of Membrane Protein Aggregates on Aggregate Size Supports a Viscous Model of Retardation of Diffusion

Cited by 79 publications

References 40 publications

Fluorescence Resonance Energy Transfer Analysis of Cytochromes P450 2C2 and 2E1 Molecular Interactions in Living Cells

Fluorescence Resonance Energy Transfer Analysis of Cytochromes P450 2C2 and 2E1 Molecular Interactions in Living Cells

A Molecular Clutch between the Actin Flow and N-Cadherin Adhesions Drives Growth Cone Migration

Variation of the Lateral Mobility of Transmembrane Peptides with Hydrophobic Mismatch

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