Testing for endocytosis in plants.

For many years endocytosis has been regarded with great scepsis by plant physiologists. Although now generally accepted, care must still be taken with experiments designed to demonstrate endocytic uptake at the plasma membrane. We have taken a critical look at the various agents which are in use as markers for plant endocytosis, pointing out pitfalls and precautions which should be taken. We also take this opportunity to introduce the tyrphostins--tyrosine kinase inhibitors--, which also seem to prevent endocytosis in plants.

Uptake of endocytic markers by rice cells: variations related to the growth phase.

Endocytosis is now considered a basic cellular process common to plant cells. Although both non-specific and receptor-mediated endocytosis appear to take place in plant cells, the physiological role of the latter remains unclear. We have investigated the endocytic process in rice cell suspensions using two biotinylated proteins, peroxidase and bovine serum albumin (bHRP and bBSA), as markers. First, we show that markers are internalized by rice cells and appear in intracellular membranes. The uptake of the two markers is temperature dependent, saturable with time and markers dose and it is competed by free biotin. Thus, it shows the properties of a receptor-mediated process. We also show that uptake of markers is strongly influenced by growth phase as optimal uptake occurs during the lag phase, but the initiation of the exponential growth phase decreases uptake drastically. Arrest of the cell cycle by starvation of either a nutrient (phosphate) or a growth regulator (2,4-dichlorophenoxyacetic acid), both components of the culture medium, does not modify the rate of bBSA uptake. Subsequent readdition of these components results in growth recovery and a dramatic decrease in bBSA uptake. On the other hand, nocodazole treatment, a method to arrest the cell cycle by microtubule depolymerization, inhibited bBSA uptake. The possible causes for this arrest of endocytosis are discussed.

Characterization of Cop I coat proteins in plant cells.

Membrane traffic in eukaryotic cells is mediated by COP (coat protein)-coated vesicles. Their existence in plant cells has not yet been unequivocally demonstrated, although coated vesicles (probably with a COP coat) can be seen by electron microscopy. At the gene level, plant cells seem to contain all the components necessary to form COP-coated vesicles. In this paper, we have used antibodies raised against mammalian COPI coat proteins to detect putative homologues in rice (Oryza sativa) cells. Using these antibodies, we have found that rice cells contain alpha-, beta-, beta'-, and gamma-COP, as well as ADP-ribosylation factor (ARF) 1 protein. In addition, we show that antibodies against mammalian beta'-COP can immunoprecipitate not only beta'-COP but also alpha-, beta-, and gamma-COP, suggesting that COPI components in rice cells exist as a complex (or coatomer) in the cytosol, as in mammalian cells. Finally, we show that COP binding to membranes is GTP-dependent, and that ARF1 also binds to membranes in a GTP-dependent manner.

NSF is required for transport from early to late endosomes.

Protein transport between early and late endosomes is a major membrane trafficking pathway in the cell followed by many proteins, including all down-regulated receptors. Yet, little is known at the molecular level about the mechanisms regulating membrane interactions in the endocytic pathway beyond early endosomes. In this study, we have used an in vitro transport assay to study the biochemical properties of endosome docking/fusion events. Our data demonstrate that N-ethylmaleimide (NEM) sensitive factor (NSF) and its soluble associated proteins (SNAPs) are required for transport from early to late endosomes, as well as at all other steps of endosomal membrane transport. We also find that these proteins are enriched on endosomal membranes. In addition, our studies suggest that besides NSF/SNAPs, another NEM-sensitive component may also be involved in docking/fusion at this late stage of the pathway. Finally, we find that, in contrast to Golgi membranes, NSF association to both early and late endosomal membranes occurs via an ATP-independent mechanism, indicating that the binding properties of endosomal and biosynthetic NSF are different. Our data thus show that NSF/SNAPs, perhaps together with another NEM-sensitive factor, are part of the basic molecular machinery which controls docking/fusion events during transport from early to late endosomes, along the lysosomal degradation pathway.

Functional dissection of COP-I subunits in the biogenesis of multivesicular endosomes.

In the present paper, we show that transport from early to late endosomes is inhibited at the restrictive temperature in a mutant CHO cell line (ldlF) with a ts-defect in epsilon coatomer protein (epsilonCOP), although internalization and recycling continue. Early endosomes then appear like clusters of thin tubules devoid of the typical multivesicular regions, which are normally destined to become vesicular intermediates during transport to late endosomes. We also find that the in vitro formation of these vesicles from BHK donor endosomes is inhibited in cytosol prepared from ldlF cells incubated at the restrictive temperature. Although epsilonCOP is rapidly degraded in ldlF cells at the restrictive temperature, cellular amounts of the other COP-I subunits are not affected. Despite the absence of epsilonCOP, we find that a subcomplex of beta, beta', and zetaCOP is still recruited onto BHK endosomes in vitro, and this binding exhibits the characteristic properties of endosomal COPs with respect to stimulation by GTPgammaS and sensitivity to the endosomal pH. Previous studies showed that gamma and deltaCOP are not found on endosomes. However, alphaCOP, which is normally present on endosomes, is no longer recruited when epsilonCOP is missing. In contrast, all COP subunits, except obviously epsilonCOP itself, still bind BHK biosynthetic membranes in a pH-independent manner in vitro. Our observations thus indicate that the biogenesis of multivesicular endosomes is coupled to early endosome organization and depends on COP-I proteins. Our data also show that membrane association and function of endosomal COPs can be dissected: whereas beta, beta', and zetaCOP retain the capacity to bind endosomal membranes, COP function in transport appears to depend on the presence of alpha and/or epsilonCOP.

Acidic cytosolic proteins are preferentially imported into rat liver lysosomes.

Previous studies have reported that lysosomes isolated from human diploid fibroblasts and from rat liver can selectively import and degrade specific proteins. We have now reinvestigated this selectivity using an in vitro assay with rat liver lysosomes and an extract of cytosolic proteins prepared from cultured cells labeled to equilibriums with [35S-]methionine. Analysis by two-dimensional gel electrophoresis and autoradiography of the cytosolic proteins bound to the lysosomal membrane and imported into the lysosomes shows that when all cytosolic proteins are simultaneously present in the in vitro assay the lysosomal uptake also occurs in a specific manner. These findings suggest that isolated lysosomes are able to discriminate among different proteins, selecting those with certain features for lysosomal degradation. Additional characterization of the cytosolic proteins which are selectively imported by lysosomes shows that a common structural feature of most, but not all, of these proteins is an acidic isoelectric point (pI <6.0) and a small or intermediate size. This observation is in agreement with earlier studies which established a relationship between the in vivo half-lives of cytosolic proteins in rat liver and their net charge, with acidic proteins, in general, being degraded more rapidly than neutral or basic proteins. The reasons for this preference are still uncertain, although a possible explanation is presented.

Selective uptake and degradation of c-Fos and v-Fos by rat liver lysosomes.

The transcription factor c-Fos is a short-lived protein and calpains and ubiquitin-dependent systems have been proposed to be involved in its degradation. In this report, we consider a lysosomal degradation pathway for c-Fos. Using a cell-free assay, we have found that freshly isolated lysosomes can take up and degrade c-Fos with high efficiency. v-Fos, the oncogenic counterpart of c-Fos, can also be taken up by lysosomes, yet the amount of incorporated protein is much lower. c-Fos uptake is independent of its phosphorylation state but it appears to be regulated by dimerization with differentially phosphorylated forms of c-Jun, while v-Fos escapes this regulation. Moreover, we show that c-Fos is immunologically detected in lysosomes isolated from the liver of rats treated with the protease inhibitor leupeptin. Altogether, these results suggest that lysosomes can also participate in the selective degradation of c-Fos in rat liver.

An endosomal beta COP is involved in the pH-dependent formation of transport vesicles destined for late endosomes.

In this paper, we show that beta COP is present on endosomes and is required for the formation of vesicles which mediate transport from early to late endosomes. Both the association of beta COP to endosomal membranes as well as transport vesicle formation depend on the lumenal pH. We find that epsilon COP, but not gamma COP, is also associated to endosomes, and that this association is also lumenal pH dependent. Our data, thus, indicate that a subset of COPs is part of the mechanism regulating endosomal membrane transport, and that membrane association of these COPs is controlled by the acidic properties of early endosomes, presumably via a trans-membrane pH sensor.

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.

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.

Uptake and degradation of glyceraldehyde-3-phosphate dehydrogenase by rat liver lysosomes.

The molecular mechanisms involved in the degradation of individual cellular proteins are probably unique and characteristic. We have investigated in rat liver the degradation of glyceraldehyde-3-phosphate dehydrogenase, an abundant cytosolic enzyme of the glycolytic pathway. Immunoblot analysis of isolated liver lysosomes from rats treated with lysosomal inhibitors show that this protein is degraded, at least in part, by a lysosomal pathway. This pathway was further investigated by incubating the enzyme with lysosomes in a cell-free system, followed by proteolysis measurements, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of lysosomes, and electron microscopic immunocytochemistry. We postulate that the degradative mechanism of glyceraldehyde-3-phosphate dehydrogenase includes a temperature-dependent lysosomal pathway, different from classical nonspecific macroautophagy. The postulated pathway involves: binding of the enzyme to the lysosomal membrane, entry into the lysosomal matrix, and degradation. This cell-free system, which can also incorporate in vitro synthesized proteins, should allow further advances toward clarifying the complex signals that regulate protein degradation as well as its close interrelationship with protein synthesis.

Effects of centrifugation on the degradation of short-lived proteins in exponentially growing cultured cells.

The degradation mechanisms of short-lived proteins in cultured cells are unknown, probably due to the lack of procedures which specifically affect the degradation of these proteins. We found that centrifugation of cultured cells, growing either in monolayer or in suspension, between 5000 and 25,000g for 30 min, inhibits (more than 50%) the degradation of short-lived proteins but not of long-lived proteins. Protein synthesis or cell viability is not affected. Centrifugation also disorganizes the Golgi apparatus, as checked by routine electron microscopy, and inhibits the degradation of endocytosed proteins (a lysosomal process which is controlled by the Golgi apparatus). Using different centrifugation speeds, a good correlation was found between alteration of the Golgi apparatus and inhibition of protein degradation.

Vanadate inhibits degradation of short-lived, but not of long-lived, proteins in L-132 human cells.

Vanadate, at concentrations higher than 0.04 mM, inhibits the intracellular degradation of short-lived proteins in exponentially growing L-132 human cells. The inhibition is not due to a decrease in viability or in the ATP contents of the cells. Since vanadate decreases proteolysis in cell extracts, the inhibition appears to affect the proteinases which degrade these proteins. Under optimal nutritional conditions, the degradation of long-lived proteins is accelerated by vanadate, thus providing additional evidence that in exponentially growing cultured cells degradation of short- and long-lived proteins occurs by different processes. Vanadate also efficiently inhibits the lysosomal degradation of endocytosed proteins and of long-lived proteins under step-down conditions. However, this effect seems to be unrelated to the observed inhibition of degradation of short-lived proteins, because chloroquine and leupeptin, which inhibit degradation of proteins by lysosomes, do not modify the degradation of these proteins. Our results provide for the first time a probe which, owing to its opposite effects on the degradation of short- and long-lived proteins, could be useful to clarify the mechanisms involved in protein degradation in cultured cells.

ATP and 2,3-bisphosphoglycerate: models of metabolites for the regulation of intracellular protein degradation.

The main question in protein turnover is what determines the susceptibility of a given protein molecule to proteolytic degradation. Much evidence supports a role for the structural characteristics of individual proteins in determining their specific degradation rates. However, changes in the environment can influence these characteristics and thus the degradation rates. Since intracellular proteins in vivo are in a natural environment, substrates, products, cofactors and other low molecular weight compounds are often bound to the proteins, and probably contribute thereby to the vastly different half-lives of proteins. This paper reviews recent results from the authors' laboratory on the possible regulation of intracellular protein degradation by low molecular weight components. We have centered our studies on 2,3-bisphosphoglycerate and ATP, which modify, in opposite directions, the proteolytic susceptibility of specific mitochondrial and cytosolic proteins to lysosomal and non-lysosomal proteases. As shown, these metabolites can also modify the microautophagic uptake of certain proteins as well as the degradation rate of proteins in cultured cells.

The mitochondrial probe rhodamine 123 inhibits in isolated hepatocytes the degradation of short-lived proteins.

The fluorescent dye rhodamine 123 (R123) decreases the intracellular ATP levels and also inhibits the degradation of short-lived proteins in isolated hepatocytes. This inhibition affects lysosomal and, to some extent, non-lysosomal mechanisms. The degradation of short-lived proteins decreases more when ATP levels are less than 40% of those in control cells, in contrast to the reported linear correlation between ATP levels and degradation of long-lived proteins. R123 provides a powerful probe for clarifying the proteolytic mechanisms involved in degradation of short-lived proteins and the ATP requirements in protein degradation. Indeed, as illustrated, the results suggest different mechanisms for the degradation of short- and long-lived proteins. Moreover, they provide a warning for the clinical use of this reagent.

Effects of inhibition of ornithine aminotransferase or of general aminotransferases on urea and citrulline synthesis and on the levels of acetylglutamate in isolated rat hepatocytes.

Canaline and gabaculine, inhibitors of gamma-aminotransferases and thus of ornithine aminotransferase (E.C. 2.6.1.13), decreased the flow through ornithine carbamoyl transferase (E.C. 2.1.3.3) in isolated rat hepatocytes incubated with 10 mM NH4Cl and ornithine. The levels of acetylglutamate, an essential activator of carbamoyl phosphate synthetase (ammonia) (E.C. 6.3.4.16), were also decreased, suggesting that the inhibitors had also caused a decrease in the rate of carbamoyl phosphate synthesis. Under these conditions, ornithine appears to be a precursor of acetylglutamate, via ornithine aminotransferase, possibly as a consequence of glutamate synthesis. The influence of aminooxyacetate, an aminotransferase inhibitor, has also been examined.

2,3-Bisphosphoglycerate inhibits ATP-stimulated proteolysis.

Intracellular protein breakdown could be regulated at the substrate level by changes in the environment. Under in vitro conditions, ATP increases the proteolytic susceptibility of several mitochondrial and cytosolic proteins, while 2,3-bisphosphoglycerate not only has the opposite effect but also prevents the ATP-stimulated proteolysis. ATP and 2,3-bisphosphoglycerate, present at relatively high levels in many tissues, provide a good model of environmental components that may influence intracellular proteolysis.