Membrane transport from early to late endosomes.
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Animals↗
Biomedical subjects
J Gruenberg
Publications and source records attributed to J Gruenberg.
Inhibition of rab5 GTPase activity stimulates membrane fusion in endocytosis.
Small GTPases of the rab family control distinct steps of intracellular transport. The function of their GTPase activity is not completely understood. To investigate the role of the nucleotide state of rab5 in the early endocytic pathway, the effects of two mutants with opposing biochemical properties were tested. The Q79L mutant of rab5, analogous with the activating Q61L mutant of p21-ras, was found to have a strongly decreased intrinsic GTPase activity and was, unlike wild-type rab5, found mainly in the GTP-bound form in vivo. Expression of this protein in BHK and HeLa cells led to a dramatic change in cell morphology, with the appearance of unusually large early endocytic structures, considerably larger than those formed upon overexpression of wild-type rab5. An increased rate of transferrin internalization was observed in these cells, whereas recycling was inhibited. Cytosol containing rab5 Q79L stimulated homotypic early endosome fusion in vitro, even though it contained only a small amount of the isoprenylated protein. A different mutant, rab5 S34N, was found, like the inhibitory p21-ras S17N mutant, to have a preferential affinity for GDP. Overexpression of rab5 S34N induced the accumulation of very small endocytic profile and inhibited transferrin endocytosis. This protein inhibited fusion between early endosomes in vitro. The opposite effects of the rab5 Q79L and S34N mutants suggest that rab5:GTP is required prior to membrane fusion, whereas GTP hydrolysis by rab5 occurs after membrane fusion and functions to inactivate the protein.
Animals↗
Vacuolar ATPase activity is required for endosomal carrier vesicle formation.
A proton pump, the vacuolar ATPase, is known to generate the acidic lumenal environment of endosomes and lysosomes. We have investigated the role of the vacuolar ATPase in endocytic membrane traffic by combining electron microscopy in vivo with a cell-free assay that reconstitutes endosome fusion in vitro. Our observations show that inactivation of this proton pump with bafilomycin A1 has no significant effects on internalization or recycling back to the plasma membrane. However, early endosomes become highly tubular and endocytosed markers do not appear in late endosomes. Our data strongly suggest that, upon inactivation of the proton pump, the formation of a vesicular intermediate between early and late endosomes, which we term endosomal carrier vesicle, is impaired.
Adenosine Triphosphatases↗
The N-terminal domain of a rab protein is involved in membrane-membrane recognition and/or fusion.
Proteins of the YPT1/SEC4/rab family are well documented to be involved in the regulation of membrane transport. We have previously reported that rab5 regulates endosome-endosome recognition and/or fusion in vitro. Here, we show that this process depends on the rab5 N-terminal domain. Treatment of early endosomal membranes at a low trypsin concentration essentially abolished fusion and cleaved rab5 to a 1 kDa smaller polypeptide. Two-dimensional gel analysis suggested that rab5 is one of the few, if not the only, polypeptides cleaved by trypsin under these conditions. Whereas endosome fusion could be stimulated by cytosol prepared from cells overexpressing rab5 (and thus containing high amounts of the protein), this stimulation was abolished by trypsin-treatment of the cytosol. Trypsin-treated cytosol prepared from mock-transfected cells, which contains very low amounts of rab5, showed no inhibitory activity indicating that rab5 is the target of trypsin in these experiments. Purified rab5 prepared after expression in Escherichia coli was treated with trypsin, which cleaved the protein at the N-terminus. A synthetic peptide of rab5 N-terminal domain inhibited endosome fusion in our cell-free assay. A version of the same peptide truncated at the N-terminus or a peptide of rab3 N-terminal domain were without effects. Altogether, these observations suggest that the N-terminal domain of rab5 is involved in the process of early endosome recognition and/or fusion, presumably because it interacts with another component of the transport machinery.
Amino Acid Sequence↗
Isoproterenol-induced redistribution of endosomes in cardiac myocytes.
During myocardial infarction, it is well known that the cytoplasm of cardiac myocytes becomes more acidic due to lactate accumulation. The resulting necrosis is believed to result, at least in part, from the leakage of lysosomal enzymes into the cytoplasm. In addition, it has previously been shown that cytoplasm acidification in tissue culture cells can cause the redistribution of late endosomes and tubular lysosomes. In the present study, we have investigated whether lysosomal/endosomal structures were affected during isoproterenol-induced infarct-like myocardial necrosis in rat heart in vivo. In parallel, we treated rat primary myocyte cultures with a high dose of isoproterenol or low pH. We followed the fate of lysosomal enzymes, a lysosomal membrane glycoprotein and the cation-independent mannose 6-phosphate receptor. Lysosomes were intact until irreversible injury became evident suggesting that the lysosomal enzyme release concomitant with the leakage of cytoplasmic enzymes is merely a consequence of cell death. During the early phase of injury, when the myocyte cytoplasm was mildly acidified, late endosomes showed fragmentation and microtubule-dependent movement towards the periphery, while the subcellular distribution of lysosomes was unchanged. Both processes were also observed after mild artificial acidification of the cytoplasm. Our data show that late endosomes and lysosomal trafficking are affected early during isoproterenol-induced myocardial injury causing a pH-dependent redistribution of late endosomes.
Animals↗
Phosphorylation of GDI and membrane cycling of rab proteins.
Membrane transport is known to be regulated by protein phosphorylation and by small GTPases of the rab family. Using specific antibodies, we have identified a 55 kDa phosphorylated protein which co-immunoprecipitated with the cytosolic forms of rab5 and other rab proteins. We demonstrate, on the basis of its mobility in two-dimensional electrophoresis gels and its immunological properties, that this protein is rab GDI (p55/GDI). We also found that, a minor fraction of p55/GDI is membrane associated, but, whilst also complexed with rab proteins, it is not phosphorylated. On the basis of these data we suggest that the cycling of rab proteins between membranes and cytosol is regulated by phosphorylation of p55/GDI.
Amino Acid Sequence↗
Annexins in membrane traffic.
Annexins have long been though to be involved in exocytosis, possibly by helping to create close interactions between membranes destined to undergo fusion. In this article, we examine recent observations that implicate annexins in three different steps of the endocytic pathway, suggesting that annexins may be universal modulators of membrane trafficking.
Journal Article↗
Annexin II is a major component of fusogenic endosomal vesicles.
We have used an in vitro assay to follow the proteins transferred from a donor to an acceptor upon fusion of early endosomes. The acceptor was a purified early endosomal fraction immunoisolated on beads and the donor was a metabolically-labeled early endosomal fraction in suspension. In the assay, both fractions were mixed in the presence of unlabeled cytosol, and then the beads were retrieved and washed. The donor proteins transferred to the acceptor were identified by two-dimensional gel electrophoresis and autoradiography. Approximately 50 major proteins were transferred and this transfer fulfilled all criteria established for endosome fusion in vitro. However, only a small subset of proteins was efficiently transferred, if donor endosomes were briefly sonicated to generate small (0.1 micron diam) vesicles before the assay. These include two acidic membrane proteins, and three alkaline peripheral proteins exposed on the cytoplasmic face of the membrane. Partial sequencing and Western blotting indicated that one of the latter components is annexin II, a protein known to mediate membrane-membrane interactions. Immunogold labeling of cryosections confirmed that annexin II is present on early endosomes in vivo. These data demonstrate that annexin II, together with the other four proteins we have identified, is a major component of fusogenic endosomal vesicles, suggesting that these proteins are involved in the binding and/or fusion process.
Amino Acid Sequence↗
Cytoplasmic dynein-dependent vesicular transport from early to late endosomes.
We have used an in vitro fusion assay to study the mechanisms of transport from early to late endosomes. Our data show that the late endosomes share with the early endosomes a high capacity to undergo homotypic fusion in vitro. However, direct fusion of early with late endosomes does not occur. We have purified vesicles which are intermediates during transport from early to late endosomes in vivo, and analyzed their protein composition in two-dimensional gels. In contrast to either early or late endosomes, these vesicles do not appear to contain unique proteins. Moreover, these vesicles undergo fusion with late endosomes in vitro, but not with each other or back with early endosomes. In vitro, fusion of these endosomal vesicles with late endosomes is stimulated by polymerized microtubules, consistent with the known role of microtubules during early to late endosome transport in vivo. In contrast, homotypic fusion of early or late endosomes is microtubule-independent. Finally, this stimulation by microtubules depends on microtubule-associated proteins and requires the presence of the minus-end directed motor cytoplasmic dynein, but not the plus-end directed motor kinesin, in agreement with the microtubule organization in vivo. Our data strongly suggest that early and late endosomes are separate, highly dynamic organelles, which are connected by a microtubule-dependent vesicular transport step.
Animals↗
Interactions of rab5 with cytosolic proteins.
Rab proteins, one of the subfamilies of ras-like small GTP-binding proteins, are attached to cellular compartments or transport vesicles and may determine the specificity of fusion between these compartments and vesicles. It has been proposed that they alternate between a membrane-bound and a cytosolic state during their functional cycle. We have used a photo-crosslinking approach to identify their cytosolic interaction partners. In vitro synthesized rab5 was cross-linked in the presence of ATP mainly to three cytosolic proteins of 52, 65, and 85 kDa. Sucrose density gradient centrifugation of the cross-linked products suggested that they were part of a 10-14 S complex. Furthermore, rab5 was cross-linked to these and additional cytosolic proteins of 42, 48, and 160 kDa in the absence of ATP. Unexpectedly, upon ATP depletion of the cytosol cross-linked and noncross-linked rab5 was found in a sedimentable high molecular weight structure. Other members of the rab subfamily, but not N-ras, also sedimented under these conditions. Electrophoretic and electron microscopic analysis of the pelleted material revealed that it contained actin filament bundles and intermediate filaments. Our data suggest that cytosolic rab proteins interact with several proteins in a 10-14 S complex, and that the rab proteins may interact directly or indirectly via this complex with the cytoskeleton.
Actins↗
Inhibition of membrane fusion in vitro via cyclin B but not cyclin A.
It is now clear that complexes of cdc2 kinase with "mitotic" cyclins regulate the transition between the G2 phase of the cell cycle and mitosis and that membrane traffic in mammalian cells is arrested during mitosis. Using a cell-free assay, we have previously reported that the fusion of early endosomes is, in fact, inhibited via the cdc2 kinase (Tuomikoski, T., Felix, M.-A., Dorée, M., and Gruenberg, J. (1989) Nature 342, 942-945). In the present paper, we show that this in vitro inhibition occurs efficiently only when the kinase activity is specifically evoked by a cyclin of the B-type but not by cyclins of the A-type. In addition, high resolution two-dimensional gel analysis revealed that the kinases associated with A- and B-type cyclins exhibit different substrate preferences. These data suggest that the complexes of the cdc2 kinase with different cyclins may control specific events of the cell cycle.
Animals↗
Regulation of intracellular membrane transport.
A number of proteins that are necessary for membrane transport have been identified using cell-free assays and yeast genetics. Although our knowledge of transport mechanisms remains limited, common themes are clearly emerging. In particular, specific GTP-binding proteins appear to be involved, not only at all steps of membrane traffic but also at more than one check-point within each step. The ordered sequence of events occurring during vesicle formation, targeting and fusion may be regulated in a stepwise manner by specific GTP-dependent switches, which act as modular elements of the transport mechanism.
Animals↗
A pilot study of the relationship between experts' ratings and scores generated by the NBME's Computer-Based Examination System.
This pilot study evaluates the consistency of experts' ratings of students' performances on the National Board of Medical Examiners' Computer Based Examination (CBX) cases and the relationship of those ratings to the CBX's scoring algorithm. The authors were investigating whether an automated scoring algorithm can adequately assess an examinee's management of a computer-simulated patient. In 1989-90, at the Michigan State University College of Human Medicine, eight students, completing a surgery clerkship, each managed eight CBX cases and took a computer-administered, multiple-choice examination. Six clerkship coordinators rated the students' performances in terms of overall management, efficiency, and dangerous actions. The ratings correlated highly with scores produced by the CBX's scoring system.
Algorithms↗
Plasticity of early endosomes.
We observed that the structural organization of early endosomes was significantly modified after cell surface biotinylation followed by incubation in the presence of low concentrations of avidin. Under these conditions early endosomes increased in size to form structures which extended over several micrometers and which had an intra-luminal content with a characteristic electron-dense appearance. The modified early endosomes were not formed when either avidin or biotinylation was omitted, suggesting that they resulted from the cross-linking of internalized biotinylated proteins by avidin. Accumulation of a fluid-phase tracer was increased after the avidin-biotin treatment (145% after 45 min). Both recycling and transport to the late endosomes still occurred, albeit to a somewhat lower extent than in control cells. Quantitative electron microscopy showed that the volume of the endosomal compartment was increased approximately 1.5-fold but that the surface area of the compartment decreased relative to its volume after avidin-biotin treatment. Finally, overexpression of a rab5 mutant, which is known to inhibit early endosome fusion in vitro, prevented the formation of these structures in vivo and caused early endosome fragmentation. Altogether, our data suggest that early endosomes exhibit a high plasticity in vivo. Cross-linking appears to interfere with this dynamic process but does not arrest membrane traffic to/from early endosomes.
Animals↗
Hypervariable C-terminal domain of rab proteins acts as a targeting signal.
Mammalian cells express many ras-like low molecular mass GTP-binding proteins (rab proteins) that are highly homologous to the Ypt1 and Sec4 proteins involved in controlling secretion in yeast. Owing to their structural similarity and to their variety, rab proteins have been postulated to act as specific regulators of membrane traffic in exocytosis and endocytosis, and rab5 has been shown to be involved in early endosome fusion in vitro. In agreement with their postulated functions, all rab proteins studied so far have been found in distinct subcompartments along the exocytic or endocytic pathways. To define the region mediating their specific localization, we transiently expressed rab2, rab5 and rab7 hybrid proteins in BHK cells, and determined their intracellular localization by immunofluorescence confocal microscopy and subcellular fractionation. Here we present evidence that the highly variable C-terminal domain contains structural elements necessary for the association of rab proteins with their specific target membranes in the endocytic pathway.
Animals↗
rab5 controls early endosome fusion in vitro.
The small GTP-binding protein rab5 was previously localized on early endosomes and on the cytoplasmic face of the plasma membrane. Using a cell-free assay, we have now tested whether rab5 is involved in controlling an early endocytic fusion event. Fusion could be inhibited by cytosol containing the overexpressed mutant rab5lle133, which does not bind GTP on blots, and by antibodies against rab5, but not against rab2 or rab7. In contrast, fusion was stimulated with cytosol containing overexpressed wild-type rab5. Cytosols containing high levels of rab2 or mutant rab5 with the 9 carboxy-terminal amino acids deleted, which bind GTP on blots, had no effects. Finally, the inhibition mediated by anti-rab5 antibodies could be overcome by complementing the assay with the cytosol containing wild-type rab5, but not with the same cytosol depleted of rab5, nor with cytosol containing the rab5 mutants or rab2. These in vitro findings strongly suggest that rab5 is involved in the process of early endosome fusion.
Animals↗
In vitro studies of endocytic membrane traffic.
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Biological Transport↗
The arguments for pre-existing early and late endosomes.
The past decade has seen the elucidation of many of the events and processes responsible for receptor-mediated endocytosis. However, a fundamental question about the endocytic pathway remains unresolved: do early endosomes mature into late endosomes, or are these two distinct and pre-existing cellular organelles? General opinion tends to favour the former possibility, to the point where one poster session at the recent American Society for Cell Biology meeting was entitled 'Maturation of Endosomes'. This article draws together new data arguing in favour of pre-existing early and late endosomes, between which transport occurs by vesicle budding and fusion.
Journal Article↗