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Ectoenzymes of the kidney microvillar membrane. Differential solubilization by detergents can predict a glycosyl-phosphatidylinositol membrane anchor.

ABSTRACT: The pattern of solubilization of nine kidney microvillar ectoenzymes by a range of detergents distinguished two classes of membrane proteins: those released from the membrane by bacterial phosphatidylinositol-specific phospholipase C and those not so released. The latter group of transmembrane proteins were solubilized efficiently (greater than 80%) by all the detergents examined. In contrast, proteins released by phosphatidylinositol-specific phospholipase C were solubilized effectively only by octyl glucoside, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonate and sodium deoxycholate. Octyl glucoside solubilized the amphipathic forms of the ectoenzymes examined, suggesting that this may be a useful detergent in the purification of glycosyl-phosphatidylinositol-anchored ectoenzymes.

SUBMITTER: Hooper NM

PROVIDER: S-EPMC1148935 | biostudies-other | 1988 Mar

REPOSITORIES: biostudies-other

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Project description:Glycosylphosphatidylinositol (GPI) is a post-translational modification resulting in the attachment of modified proteins to the outer leaflet of the plasma membrane. Tissue culture experiments have shown GPI-anchored proteins (GPI-APs) to be targeted to the apical membrane of epithelial cells. However, the in vivo importance of this targeting has not been investigated since null mutations in GPI biosynthesis enzymes in mice result in very early embryonic lethality. Missense mutations in the human GPI biosynthesis enzyme pigv are associated with a multiple congenital malformation syndrome with a high frequency of Hirschsprung disease and renal anomalies. However, it is currently unknown how these phenotypes are linked to PIGV function. Here, we identify a temperature-sensitive hypomorphic allele of PIGV in Caenorhabditis elegans, pigv-1(qm34), enabling us to study the role of GPI-APs in development. At the restrictive temperature we found a 75% reduction in GPI-APs at the surface of embryonic cells. Consequently, ~80% of pigv-1(qm34) embryos arrested development during the elongation phase of morphogenesis, exhibiting internal cysts and/or surface ruptures. Closer examination of the defects revealed them all to be the result of breaches in epithelial tissues: cysts formed in the intestine and excretory canal, and ruptures occurred through epidermal cells, suggesting weakening of the epithelial membrane or membrane-cortex connection. Knockdown of piga-1, another GPI biosynthesis enzymes resulted in similar phenotypes. Importantly, fortifying the link between the apical membrane and actin cortex by overexpression of the ezrin/radixin/moesin ortholog ERM-1, significantly rescued cyst formation and ruptures in the pigv-1(qm34) mutant. In conclusion, we discovered GPI-APs play a critical role in maintaining the integrity of the epithelial tissues, allowing them to withstand the pressure and stresses of morphogenesis. Our findings may help to explain some of the phenotypes observed in human syndromes associated with pigv mutations.

Project description:Understanding the pathways of solubilization of lipid membranes is of high importance for their use in biotechnology and industrial applications. Although lipid vesicle solubilization by classical detergents has been widely investigated, there are few systematic structural and kinetic studies where different detergents are compared under varying conditions. This study used small-angle X-ray scattering to determine the structures of lipid/detergent aggregates at different ratios and temperatures and studied the solubilization in time using the stopped-flow technique. Membranes composed of either of two zwitterionic lipids, DMPC or DPPC, and their interactions with three different detergents, sodium dodecyl sulfate (SDS), n-dodecyl-beta-maltoside (DDM), and Triton X-100 (TX-100), were tested. The detergent TX-100 can cause the formation of collapsed vesicles with a rippled bilayer structure that is highly resistant to TX-100 insertion at low temperatures, while at higher temperatures, it partitions and leads to the restructuring of vesicles. DDM also causes this restructuring into multilamellar structures at subsolubilizing concentrations. In contrast, partitioning of SDS does not alter the vesicle structure below the saturation limit. Solubilization is more efficient in the gel phase for TX-100 but only if the cohesive energy of the bilayer does not prevent sufficient partitioning of the detergent. DDM and SDS show less temperature dependence compared to TX-100. Kinetic measurements reveal that solubilization of DPPC largely occurs through a slow extraction of lipids, whereas DMPC solubilization is dominated by fast and burst-like solubilization of the vesicles. The final structures obtained seem to preferentially be discoidal micelles where the detergent can distribute in excess along the rim of the disc, although we do observe the formation of worm- and rodlike micelles in the case of solubilization of DDM. Our results are in line with the suggested theory that bilayer rigidity is the main factor influencing which aggregate is formed.

Project description:It is difficult to maintain a target membrane protein in a soluble and functional form in aqueous solution without biological membranes. Use of surfactants can improve solubility, but it remains challenging to identify adequate surfactants that can improve solubility without damaging their native structures and biological functions. Here we report the use of a new class of lipopeptides to solubilize photosystem I (PS-I), a well known membrane protein complex. Changes in the molecular structure of these surfactants affected their amphiphilicity and the goal of this work was to exploit a delicate balance between detergency and biomimetic performance in PS-I solubilization via their binding capacity. Meanwhile, the effects of these surfactants on the thermal and structural stability and functionality of PS-I in aqueous solution were investigated by circular dichroism, fluorescence spectroscopy, SDS-PAGE analysis and O2 uptake measurements, respectively. Our studies showed that the solubility of PS-I depended on both the polarity and charge in the hydrophilic head of the lipopeptides and the length of its hydrophobic tail. The best performing lipopeptides in favour of PS-I solubility turned out to be C14DK and C16DK, which were comparable to the optimal amphiphilicity of the conventional chemical surfactants tested. Lipopeptides showed obvious advantages in enhancing PS-I thermostability over sugar surfactant DDM and some full peptide amphiphiles reported previously. Fluorescence spectroscopy along with SDS-PAGE analysis demonstrated that lipopeptides did not undermine the polypeptide composition and conformation of PS-I after solubilization; instead they showed better performance in improving the structural stability and integrity of this multi-subunit membrane protein than conventional detergents. Furthermore, O2 uptake measurements indicated that PS-I solubilized with lipopeptides maintained its functionality. The underlying mechanism for the favorable actions of lipopeptide in PS-I solubilization and stabilization is discussed.

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Ectoenzymes of the kidney microvillar membrane. Differential solubilization by detergents can predict a glycosyl-phosphatidylinositol membrane anchor.

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