Alkyl glucosides as effective solubilizing agents for bovine rhodopsin. A comparison with several commonly used detergents.

The suitability of octyl and decyl-beta-D-glucoside as solubilizing agents for the bovine retinal rod outer segment disc membrane was investigated and compared to that of hexadecyltrimethylammonium bromide, N,N-dimethyldodecylamine oxide, Emulphogene BC-720 and digitonin. The properties measured included the thermal stability of rhodopsin, regenerability of bleached rhodopsin by addition of 11-cis-retinal, and the rate of denaturation of bleached rhodopsin as measured by changes in the ultraviolet CD spectrum. Denaturing tendencies of the detergents were also evaluated by observing their effects on the absorption and CD spectra of sperm whale metmyoglobin. Our results demonstrate that octyl glucoside is superior to the other detergents, with the possible exception of digitonin, by the above criteria. Unlike digitonin, however, octyl glucoside affords rapid solubilization of the disc membrane and is itself highly soluble. Decyl glucoside has properties equivalent or superior to octyl glucoside, but salts and buffers interfere with its ability to solubilize the disc membrane. The well defined chemical composition, ease of removal by dialysis, and non-denaturing properties of the alkyl glucosides make them attractive detergents for membrane research.     

Binding and activation of rod outer segment phosphodiesterase and guanosine triphosphate binding protein by disc membranes: influence of reassociation method and divalent cations

Attempts to optimize the recovery of light-stimulated phosphodiesterase activity following reassociation of the hypotonically extractable proteins derived from retinal rod segments with hypotonically stripped disc membranes lead to the following observations: the best reassociations were obtained by mixing proteins and stripped disc membranes under hypotonic conditions and slowly increasing the salt concentration; the binding of G-protein and phosphodiesterase to stripped disc membrane occurs in less than 5 minutes and the recovery of light-stimulated phosphodiesterase activation in response to subsaturating stimulus levels requires 2-3 h to plateau. Stripped disc membranes and proteins were reassociated in 'isotonic' buffers containing KCl/NaCl, KCl/NaCl plus Mg2+, or KCl/NaCl plus Ca2+. Large fractional rhodopsin bleaches produced nearly identical light-stimulated phosphodiesterase activities in each of these samples and in the control rod outer segment membranes. Rod outer segment membranes and reassociated stripped disc membrane samples containing divalent cations showed similar phosphodiesterase activities in response to low fractional rhodopsin bleaches (e.g. less than or equal to 0.1%), however, samples devoid of divalent cations during reassociation required rhodopsin bleaches up to 10-fold larger to elicit comparable phosphodiesterase activities. These results suggest that not all phosphodiesterase and/or G-protein molecules bound to the disc membrane surface are equivalent with regard to their efficiency of activation by bleached rhodopsin and that divalent cations can modulate the distribution of G-protein and/or phosphodiesterase between these populations.     

Analysis of polypeptide disposition in human erythrocyte membranes employing membrane inversion

High resolution segregation of erythrocyte membrane polypeptides achieved by isoelectric focusing in 8 M urea was employed in conjunction with surface-restricted radioiodination to analyze the disposition of polypeptides within the human erythrocyte membrane. Several membrane polypeptides showed significant uptake of radioiodine, with the principal labeled component migrating between pH values of 3.0 and 3.5. Two approaches were taken in examining membrane polypeptide disposition on both faces of the erythrocyte membrane. Saturation labeling of the outer face of the membrane with one iodine isotope followed by cell lysis and re-iodination with a second iodine isotope did not prove feasible and another procedure based on surface iodination with ¹²⁵I, formation of sealed inside-out vesicles and re-iodination with ¹³¹I was adopted. Studies of sialic acid release from the membrane surface and trypsin cleavage of radioiodinated peptides indicated that selectively labeled, sealed inside-out vesicles had been formed. The ratio of ¹²⁵I to ¹³¹I in membrane polypeptides separated by isoelectric focusing confirmed the existence of externally disposed, internally disposed and spanning proteins.     

A Mediator of Rho-dependent Invasion Moonlights as a Methionine Salvage Enzyme

RhoA controls changes in cell morphology and invasion associated with cancer phenotypes. Cell lines derived from melanoma tumors at varying stages revealed that RhoA is selectively activated in cells of metastatic origin. We describe a functional proteomics strategy to identify proteins regulated by RhoA and report a previously uncharacterized human protein, named “mediator of RhoA-dependent invasion (MRDI),” that is induced in metastatic cells by constitutive RhoA activation and promotes cell invasion. In human melanomas, MRDI localization correlated with stage, showing nuclear localization in nevi and early stage tumors and cytoplasmic localization with plasma membrane accentuation in late stage tumors. Consistent with its role in promoting cell invasion, MRDI localized to cell protrusions and leading edge membranes in cultured cells and was required for cell motility, tyrosine phosphorylation of focal adhesion kinase, and modulation of actin stress fibers. Unexpectedly MRDI had enzymatic function as an isomerase that converts the S-adenosylmethionine catabolite 5-methylribose 1-phosphate into 5-methylribulose 1-phosphate. The enzymatic function of MRDI was required for methionine salvage from S-adenosylmethionine but distinct from its function in cell invasion. Thus, mechanisms used by signal transduction pathways to control cell movement have evolved from proteins with ancient function in amino acid metabolism.Cutaneous malignant melanoma has doubled in incidence over the past 30 years. Stages involved in progression of melanocytes to melanoma based on clinical and histopathological features include nontumorigenic nevi, dysplastic or atypical nevi, primary radial growth phase and vertical growth phase melanoma, and metastatic melanoma (1). Metastatic melanomas are often resistant to treatment; therefore therapeutic strategies require a more complete understanding of molecular determinants of this disease, particularly those involved in the invasive phenotype (2).Rho GTPases control a wide range of cellular responses including cell movement, morphogenesis, and coordinated migration (3). These pathways are implicated in malignant cell transformation and metastasis based on in vitro evidence showing tumorigenic and invasive responses to enhanced signaling in cell lines. Studies have demonstrated that overexpression of RhoC enhances invasion and metastasis in mouse xenografts of human melanoma and lung cancer cell lines (4, 5). In addition, some human tumors show elevated expression of Rho GTPases and exchange factors and/or reduced expression of GTPase-activating factors (6–8). Signaling through RhoA promotes actin polymerization and stress fiber formation, providing cells with contractile force needed for cell movement. Rho-GTP interacts with various effectors, including Rho-activated kinase, which promotes actin-myosin assembly via phosphorylation of myosin light chain phosphatase (9), or diaphanous-related formins, which nucleate actin filaments and stabilize microtubules (10, 11). Studies of cultured melanoma cells have revealed an “amoeboid” invasion mechanism involving RhoA-dependent Rho-activated kinase activation and inactivation of Rac (12, 13).RhoA also controls the formation and turnover of focal adhesion contacts, which mediate interactions between extracellular matrix and the actin cytoskeleton (14, 15). Signaling involves activation of Src and focal adhesion kinase (FAK)¹ and subsequent tyrosine phosphorylation of proteins recruited to integrin receptor complexes (16). Embryonic cells from FAK^−/− mice lose motility and cannot be rescued with FAK harboring a Y397F autophosphorylation site mutation not because they fail to form focal adhesions but because they are unable to disassemble focal adhesions (17). Thus, Rho controls cell movement by modulating the turnover of focal adhesion complexes via FAK. However, the mechanisms by which Rho GTPases control FAK are incompletely understood.In this study, we report that RhoA was constitutively activated in melanoma cells in a stage-specific pattern with elevated activity in cells from metastatic tumors. We present a functional proteomics screen for molecular targets of RhoA from which we identified a previously uncharacterized human protein induced in response to constitutive RhoA activation. This protein promoted Rho-dependent cell invasion and cell motility and provided a novel link for regulation of FAK tyrosine phosphorylation by RhoA. Thus, we refer to it as “mediator of Rho-dependent invasion (MRDI).” Although human MRDI has not been studied previously, it shows close sequence similarity to a methylthioribose-1-phosphate isomerase, which functions in methionine salvage pathways characterized in bacteria and yeast. We demonstrated that MRDI indeed has methylthioribose-1-phosphate isomerase activity and is required for methionine salvage in human cells. We further demonstrated that the catalytic activity of MRDI is independent of its role in cell invasion. Thus, MRDI is a dual function protein with promiscuous roles both as a metabolic enzyme and as an effector of signaling and cancer cell invasion.     

CRL4(Cdt2)-mediated destruction of the histone methyltransferase Set8 prevents premature chromatin compaction in S phase

The proper coordination between DNA replication and mitosis during cell-cycle progression is crucial for genomic stability. During G2 and mitosis, Set8 catalyzes monomethylation of histone H4 on lysine 20 (H4K20me1), which promotes chromatin compaction. Set8 levels decline in S phase, but why and how this occurs is unclear. Here, we show that Set8 is targeted for proteolysis in S phase and in response to DNA damage by the E3 ubiquitin ligase, CRL4(Cdt2). Set8 ubiquitylation occurs on chromatin and is coupled to DNA replication via a specific degron in Set8 that binds PCNA. Inactivation of CRL4(Cdt2) leads to Set8 stabilization and aberrant H4K20me1 accumulation in replicating cells. Transient S phase expression of a Set8 mutant lacking the degron promotes premature H4K20me1 accumulation and chromatin compaction, and triggers a checkpoint-mediated G2 arrest. Thus, CRL4(Cdt2)-dependent destruction of Set8 in S phase preserves genome stability by preventing aberrant chromatin compaction during DNA synthesis.