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M I Colombo

Publications and source records attributed to M I Colombo.

8 recordsLinked to original sources
PubMed1992

Evidence of a role for heterotrimeric GTP-binding proteins in endosome fusion.

Guanosine triphosphate (GTP)-binding proteins are required for intracellular vesicular transport. Mastoparan is a peptide component of wasp venom that increases nucleotide exchange in some classes of G alpha subunits of regulatory heterotrimeric GTP-binding proteins (G proteins). Mastoparan and other compounds that increase nucleotide exchange by G proteins inhibited endosome fusion in vitro and reversed the effects of guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S), a nonhydrolyzable GTP analog. Addition of beta gamma subunits of G proteins to the fusion assay antagonized the stimulatory effect of GTP-gamma-S, confirming the participation of G proteins. These results indicate that GTP-binding proteins are required for endosome fusion and in particular that a G protein is involved. Given the function of G proteins in signal transduction, these findings may provide insight into the mechanism by which endosomal vesicles become competent for fusion after their formation at the cell surface.

Biological Transport↗
PubMed1991

Characterization of trypsin-sensitive factor(s) required for endosome-endosome fusion.

Fusion of endosomes appears to be required at early steps of receptor-mediated endocytosis. These fusion events have been reconstituted using a cell-free assay and have been shown to require both cytosolic and membrane-associated proteins. We report here that trypsinization of endosomes completely inhibited fusion. Addition of untreated cytosol cannot restore fusion of trypsinized endosomes. However, fusion activity is restored by the addition of either untreated vesicles or a high salt extract containing peripheral membrane proteins (KE). KE contains both the membrane-associated factor(s) required for the reconstitution of fusion using trypsinized endosomes and the factors that are normally provided by the cytosol. The restorative activity of KE was sensitive to trypsin treatment or incubation at 100 degrees C, but was largely N-ethylmaleimide (NEM)-resistant. This and other criteria demonstrated that the trypsin-sensitive factor is distinct from N-ethylmaleimide-sensitive factor (NSF), an NEM-sensitive protein involved in vesicular fusion, and from other known factors that may participate in membrane fusion events. Preliminary fractionation studies indicate that the restorative activity of KE is associated with one or more high molecular weight proteins. The present study indicates that a novel trypsin-sensitive protein(s) is involved in endosome-endosome fusion. This factor is membrane-associated and is not found in an active form in cytosol as prepared.

Biological Transport↗
PubMed1991

Endosomal density shift is related to a decrease in fusion capacity.

Dinitrophenol (DNP)-beta-glucuronidase and mannosylated anti-DNP IgG, which are endocytosed by the mannose receptor and delivered to lysosomes, were previously developed as probes for examination of fusion between early endosomes in a cell-free system. In this study, these probes were found to be transported by intact cells to endocytic vesicles with heavy buoyant density at different rates, as determined by Percoll gradient fractionation of cell homogenates. There was a concomitant loss of in vitro fusion activity as the ligands moved to dense compartments. In monensin-treated cells, DNP-beta-glucuronidase was retained in a light compartment corresponding to intracellular vesicles capable of fusion in vitro. Pulse-chase studies using a DNP-derivatized transferrin-alkaline phosphatase conjugate showed that a recycling ligand was always found in light intracellular vesicles that were capable of fusion to early endosomes in vitro. In contrast to cell-free systems, intact cells sequentially labeled with DNP-beta-glucuronidase and then mannosylated anti-DNP IgG showed ligand mixing in both early and late endocytic compartments. Treatment with nocodazole or colchicine did not affect the rate of DNP-beta-glucuronidase transport to heavy vesicles in intact cells, however, the extent of ligand mixing in late endosomes was decreased by microtubule disruption. Using sequentially labeled cells split into two groups, we directly compared ligand mixing in vitro to mixing by intact cells. Fusion alone does not mediate increases in vesicle density, since DNP-beta-glucuronidase/anti-DNP IgG complexes formed in vitro were found in light vesicles, while intact cells showed immune complexes predominantly in heavy vesicles. These results suggest that the density shift is an initial step in targeting to lysosomes.

Animals↗
PubMed1989

GTP gamma S stimulation of endosome fusion suggests a role for a GTP-binding protein in the priming of vesicles before fusion.

Guanosine 5'-(3-O-thio)triphosphate (GTP gamma S), a non-hydrolyzable analogue of GTP, inhibits in vitro fusion among early endocytic vesicles in the presence of high concentrations of cytosol. In this report we show that fusion is remarkably stimulated by GTP gamma S under conditions where cytosolic components are the limiting factors for the process. The amount of cytosolic factors required for maximal fusion activity is several-fold decreased by the presence of GTP gamma S. Moreover, preincubation of vesicles in the presence of cytosol and GTP gamma S allows fusion to proceed even in the absence of cytosol. Our results indicate that a GTP-binding protein facilitates the binding of cytosolic factor(s) required for endosome fusion to the endosomal membrane and stabilizes a dilution-resistant intermediate of the fusion process.

Animals↗
PubMed1988

Properties of binding sites for chloroquine in liver lysosomal membranes.

Chloroquine (CQ) is an antimalarial and antirheumatic drug that accumulates in lysosomes. We purified liver lysosomal membranes of tritosomes from albino mice injected with Triton WR 1339. The membranes were used for the binding assay with CQ in 0.01 M Tris-HCl buffer (pH 7.4). This binding was saturable, with a KD value of 6.2 microM. To understand the nature of CQ affinity, the binding was done under conditions that alter membrane structure and composition. Changes in pH, high ionic strength, and bivalent cations reversibly decreased the binding, while the effect of non-ionic detergents was partially reversed. The cationic detergent Hyamine strongly decreased the binding, and its effect was trypsin and neuraminidase had no effect. The results indicate the existence of binding sites for CQ in liver lysosomal membranes, which were strongly affected by changes of charge in the molecules involved in the binding. The treatment with the enzymes suggests that loss of polar groups of phospholipids increases the affinity of CQ by exposing protein sites located deep in the membrane, or by permiting a closer interaction between the drug and membrane lipids. CQ lysosomotropism and other effects of CQ on the lysosomal apparatus studied by other authors may be due not only to its accumulation inside the acid milieu of the lysosomes, in the same manner as other weak bases, but also to the affinity of CQ for binding sites in the lysosomal membrane.

Ammonium Chloride↗
PubMed1985

In vivo interaction between mouse liver lysosomes and chloroquine.

Particles sedimenting at 27,000 g X 10 min (MLCQ) were separated from liver homogenates of mice injected with chloroquine (CQ). The MLCQ contained most of the drug recovered in the organ as well as 50% of the liver aryl sulphatase activity. The release of CQ from MLCQ was studied in some physicochemical conditions, and in the presence of various agents known to modify membrane composition and stability. At pH 7.4, the equilibrium between free and bound CQ depended on the dilution of the MLCQ, and the time to reach equilibrium was strongly influenced by the temperature of incubation. Several agents causing membrane disruption and lysosomal enzyme leakage, such as osmotic shock, sonication and digitonin, had little effect on the CQ release. Acid and alkaline buffers, 0.55 M KCl and 0.1% Triton X-100 caused, instead, the immediate release of most of the bound CQ. Concentrations of digitonin causing the release of aryl sulphatase activity had little effect on bound CQ, suggesting that the drug is retained in lysosomes by forces and/or structures different in nature from those retaining most of the lysosomal enzyme activity. We think that the CQ trapped in lysosomes is bound to high affinity sites in membranous structures which are particularly altered by agents known to extract peripheral proteins from biological membranes or to change the conformation of molecular structures.

Animals↗