Structural proteomics : elucidating in vivo structural dynamics of integral membrane proteins by hydroxyl radical footprinting

Integral membrane proteins with the N-out topology are inserted into membranes usually in YidC- and PMF-dependent manners. The molecular basis of the various dependencies on insertion factors is not fully understood. A model protein, Pf3-Lep, is inserted independently of both YidC and PMF, whereas the V15D mutant requires both YidC and PMF in vivo. We analyzed the mechanisms that determine the insertion factor dependency in vitro. Glycolipid MPIase was required for insertion of both proteins because MPIase depletion caused a significant defect in insertion. On the other hand, YidC depletion and PMF dissipation had no effects on Pf3-Lep insertion, whereas V15D insertion was reduced. We reconstituted (proteo)liposomes containing MPIase, YidC, and/or F0F1-ATPase. MPIase was essential for insertion of both proteins. YidC and PMF stimulated Pf3-Lep insertion as the synthesis level increased. V15D insertion was stimulated by both YidC and PMF irrespective of the synthesis level. These results indicate that charges in the N-terminal region and the synthesis level are the determinants of YidC and PMF dependencies with the interplay between MPIase, YidC, and PMF.

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Two integral membrane proteins located in the cis-middle and trans-part of the Golgi system acquire sialylated N-linked carbohydrates and display different turnovers and sensitivity to cAMP-dependent phosphorylation.

The Journal of Cell Biology ◽

10.1083/jcb.105.1.215 ◽

1987 ◽

Vol 105 (1) ◽

pp. 215-227 ◽

Cited By ~ 55

Author(s):

L Yuan ◽

J G Barriocanal ◽

J S Bonifacino ◽

I V Sandoval

Keyword(s):

Monoclonal Antibodies ◽

Sialic Acid ◽

Membrane Proteins ◽

Integral Membrane Proteins ◽

Chemical Characteristics ◽

Dibutyryl Cyclic Amp ◽

Complex Type ◽

Golgi System ◽

Different Parts

The localization and chemical characteristics of two Golgi integral membrane proteins (GIMPs) have been studied using monoclonal antibodies. The two proteins are segregated in different parts of the Golgi system and whereas GIMPc(130 kD) is located in the cis and medial cisternae, GIMPt (100 kD) is confined in the trans-most cisterna and trans-tubular network. Both GIMPs are glycoproteins that contain N- and O-linked carbohydrates. The N-linked carbohydrates were exclusively of the complex type. Although excluded from the trans-side of the Golgi system, where sialylation is believed to occur, GIMPc acquires sialic acid in both its N- and O-linked carbohydrates. Sialic acid was also detected in the N-linked carbohydrates of GIMPt. GIMPc is apparently phosphorylated in the luminal domain in vivo. Phosphorylation occurred exclusively on serine and was stimulated by dibutyryl cyclic AMP. GIMPc and GIMPt displayed half-lives of 20 and 9 h, respectively.

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Plasmalemmal proteins of cultured vascular endothelial cells exhibit apical-basal polarity: analysis by surface-selective iodination.

The Journal of Cell Biology ◽

10.1083/jcb.103.6.2389 ◽

1986 ◽

Vol 103 (6) ◽

pp. 2389-2402 ◽

Cited By ~ 63

Author(s):

W A Muller ◽

M A Gimbrone

Keyword(s):

Endothelial Cells ◽

Membrane Proteins ◽

Umbilical Vein ◽

Integral Membrane Proteins ◽

Cellular Polarity ◽

Differential Distribution ◽

Endothelial Monolayers ◽

Apical Side

Vascular endothelium in vivo appears to function as a polarized epithelium. To determine whether cellular polarity exists at the level of the plasma membrane, we have examined cultured endothelial monolayers for evidence of differential distribution of externally disposed plasmalemmal proteins at apical and basal cell surfaces. Lactoperoxidase beads were used to selectively label the apical surfaces of confluent endothelial monolayers, the total surfaces of nonenzymatically resuspended cells, and the basal surfaces of monolayers inverted on poly-L-lysine-coated coverslips, while maintaining greater than 98% viability in all samples. Comparison of the SDS PAGE radioiodination patterns obtained for each surface revealed a number of specific bands markedly enriched on either apical or basal surface. This polarized distribution involved membrane-associated as well as integral membrane proteins and was observed in several strains of bovine aortic endothelial cells, as well as in both primary and passaged human umbilical vein endothelial cells. In contrast, two morphologically nonpolarized cell types, bovine aortic smooth muscle and mouse peritoneal macrophages, did not display differential localization of integral membrane proteins. Polarized distribution of integral membrane proteins was established before the formation of a confluent monolayer. When inverted (basal-side-up) monolayers were returned to culture, the apical-side-up pattern was reexpressed within a few days. These results demonstrate that cell surface-selective expression of plasmalemmal proteins is an intrinsic property of viable endothelial cells in vitro. This apical/basal asymmetry of membrane structure may provide a basis for polarized endothelial functions in vivo.

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Fractionation of Tetrahymena ciliary membranes with triton X-114 and the identification of a ciliary membrane ATPase.

The Journal of Cell Biology ◽

10.1083/jcb.107.6.2679 ◽

1988 ◽

Vol 107 (6) ◽

pp. 2679-2688 ◽

Cited By ~ 24

Author(s):

W L Dentler

Keyword(s):

Membrane Proteins ◽

Atpase Activity ◽

Tetrahymena Thermophila ◽

High Molecular Mass ◽

Integral Membrane Proteins ◽

Matrix Proteins ◽

Membrane Atpase ◽

The Matrix ◽

Triton X

Cilia were isolated from Tetrahymena thermophila, extracted with Triton X-114, and the detergent-soluble membrane + matrix proteins separated into Triton X-114 aqueous and detergent phases. The aqueous phase polypeptides include a high molecular mass polypeptide previously identified as a membrane dynein, detergent-soluble alpha and beta tubulins, and numerous polypeptides distinct from those found in axonemes. Integral membrane proteins partition into the detergent phase and include two major polypeptides of 58 and 50 kD, a 49-kD polypeptide, and 5 polypeptides in relatively minor amounts. The major detergent phase polypeptides are PAS-positive and are phosphorylated in vivo. A membrane-associated ATPase, distinct from the dynein-like protein, partitions into the Triton X-114 detergent phase and contains nearly 20% of the total ciliary ATPase activity. The ATPase requires Mg++ or Ca++ and is not inhibited by ouabain or vanadate. This procedure provides a gentle and rapid technique to separate integral membrane proteins from those that may be peripherally associated with the matrix or membrane.

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