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J Gruenberg

Publications and source records attributed to J Gruenberg.

At least 37 records · Page 2Linked to original sources
PubMed2000

Interaction of anti-phospholipid antibodies with late endosomes of human endothelial cells.

Anti-phospholipid antibodies (APLAs) are associated with thrombosis and/or recurrent pregnancy loss. APLAs bind to anionic phospholipids directly or indirectly via a cofactor such as beta(2)-glycoprotein 1 (beta(2)GPI). The lipid target of APLA is not yet established. Recently, we observed that APLAs in vitro can bind lysobisphosphatidic acid (LBPA). The internal membranes of late endosomes are enriched in this phospholipid. The current study was undertaken to determine to what extent binding of APLA to LBPA is correlated with binding to cardiolipin and to beta(2)GPI and to determine whether patient antibodies interact with late endosomes of human umbilical vein endothelial cells (HUVECs) and thus modify the intracellular trafficking of proteins. Binding of patient immunoglobulin G (n=37) to LBPA was correlated significantly with binding to cardiolipin. Although LBPA binding was correlated to a lesser extent with beta(2)GPI binding, we observed that beta(2)GPI binds with high affinity to LBPA. Immunofluorescence studies showed that late endosomes of HUVECs contain LBPA. Patient but not control antibodies recognized late endosomes, but not cardiolipin-rich mitochondria, even when we used antibodies that were immunopurified on cardiolipin. Incubation of HUVECs with patient plasma samples immunoreactive toward LBPA resulted in an accumulation of the antibodies in late endosomes and led to a redistribution of the insulinlike growth factor 2/mannose-6-phosphate receptor from the Golgi apparatus to late endosomes. Our results suggest that LBPA is an important lipid target of APLA in HUVECs. These antibodies are internalized by the cells and accumulate in late endosomes. By modifying the intracellular trafficking of proteins, APLA could contribute to several of the proposed pathogenic mechanisms leading to the antiphospholipid syndrome.

Antibodies, Antiphospholipid↗
PubMed2000

Coupled transport of p24 family members.

Recent studies show that small trans-membrane proteins of approximately 22-24 kDa (the p24 family), which are grouped into 4 sub-families by sequence homology (p23, p24, p25 and p26), are involved in the early secretory pathway. In this study, we have investigated the mutual requirements of ectopically expressed members of the p24 family for targeting to their proper cellular destination. We find that coexpression of p23 and p24 is both necessary and sufficient for each protein to be transported to the cis-Golgi network/Golgi complex. Proteins from other subfamilies did not substitute for either p23 or p24, even after multiple coexpression. However, trafficking of the p23/p24 couple was facilitated by coexpression of proteins from other sub-families. In addition, we find that the sequence resembling an endoplasmic reticulum retrieval signal present in the cytoplasmic domain of p23 (but not p24) is dispensable. In contrast, the conserved coiled-coil region in the lumenal domain is absolutely required in both p23 and p24 for proper targeting of the p23/p24 couple. These data demonstrate that p23 and p24 must interact with each other to reach their destination, but that this strict requirement is combined with a mutual dependence amongst p24 proteins. We speculate that p24 proteins can form different oligomeric complexes, which contribute to confer specialized sorting/trafficking properties to membranes of the early secretory pathway, perhaps serving as membrane organizers.

Amino Acid Sequence↗
PubMed2000

The transmembrane protein p23 contributes to the organization of the Golgi apparatus.

In previous studies we have shown that p23, a member of the p24-family of small transmembrane proteins, is highly abundant in membranes of the cis-Golgi network (CGN), and is involved in sorting/trafficking in the early secretory pathway. In the present study, we have further investigated the role of p23 after ectopic expression. We found that ectopically expressed p23 folded and oligomerized properly, even after overexpression. However, in contrast to endogenous p23, exogenous p23 molecules did not localize to the CGN, but induced a significant expansion of characteristic smooth ER membranes, where they accumulated in high amounts. This ER-derived, p23-rich subdomain displayed a highly regular morphology, consisting of tubules and/or cisternae of constant diameter, which were reminiscent of the CGN membranes containing p23 in control cells. The expression of exogenous p23 also led to the specific relocalization of endogenous p23, but not of other proteins, to these specialized ER-derived membranes. Relocalization of p23 modified the ultrastructure of the CGN and Golgi membranes, but did not affect anterograde and retrograde transport reactions to any significant extent. We conclude (i) that p23 has a morphogenic activity that contributes to the morphology of CGN-membranes; and (ii) that the presence of p23 in the CGN is necessary for the proper organization of the Golgi apparatus.

Golgi Apparatus↗
PubMed1999

Biogenesis of transport intermediates in the endocytic pathway.

Evidence is accumulating that membrane traffic between organelles can be achieved by different types of intermediates. Small (< 100 nm) and short-lived vesicles mediate transport from the plasma membrane or the trans-Golgi network to endosomes, and formation of these vesicles depends on specific adapter complexes. In contrast, transport from early to late endosomes is achieved by relatively large (approximately 0.5 microm), long-lived and multivesicular intermediates, and their biogenesis depends on endosomal COP-I proteins. Here, we review recent work on the formation of these different transport intermediates, and we discuss, in particular, coat proteins, sorting signals contained in cargo molecules and the emerging role of lipid in vesicle biogenesis.

Animals↗
PubMed1999

Nef-induced CD4 degradation: a diacidic-based motif in Nef functions as a lysosomal targeting signal through the binding of beta-COP in endosomes.

The Nef protein of primate lentiviruses downregulates the cell surface expression of CD4 through a two-step process. First, Nef connects the cytoplasmic tail of CD4 with adaptor protein complexes (AP), thereby inducing the formation of CD4-specific clathrin-coated pits that rapidly endocytose the viral receptor. Second, Nef targets internalized CD4 molecules for degradation. Here we show that Nef accomplishes this second task by acting as a connector between CD4 and the beta subunit of COPI coatomers in endosomes. A sequence encompassing a critical acidic dipeptide, located nearby but distinct from the AP-binding determinant of HIV-1 Nef, is responsible for beta-COP recruitment and for routing to lysosomes. A novel class of endosomal sorting motif, based on acidic residues, is thus revealed, and beta-COP is identified as its downstream partner.

Amino Acid Sequence↗
PubMed1999

Late endosomal membranes rich in lysobisphosphatidic acid regulate cholesterol transport.

The fate of free cholesterol released after endocytosis of low-density lipoproteins remains obscure. Here we report that late endosomes have a pivotal role in intracellular cholesterol transport. We find that in the genetic disease Niemann-Pick type C (NPC), and in drug-treated cells that mimic NPC, cholesterol accumulates in late endosomes and sorting of the lysosomal enzyme receptor is impaired. Our results show that the characteristic network of lysobisphosphatidic acid-rich membranes contained within multivesicular late endosomes regulates cholesterol transport, presumably by acting as a collection and distribution device. The results also suggest that similar endosomal defects accompany the anti-phospholipid syndrome and NPC.

Animals↗
PubMed1998

A lipid associated with the antiphospholipid syndrome regulates endosome structure and function.

Little is known about the structure and function of membrane domains in the vacuolar apparatus of animal cells. A unique feature of late endosomes, which are part of the pathway that leads to lysosomes, is that they contain a complex system of poorly characterized internal membranes in their lumen. These endosomes are therefore known as multivesicular or multilamellar organelles. Some proteins distribute preferentially within these internal membranes, whereas others are exclusively localized to the organelle's limiting membrane. The composition and function of this membrane system are poorly understood. Here we show that these internal membranes contain large amounts of a unique lipid, and thus form specialized domains within endosomes. These specialized domains are involved in sorting the multifunctional receptor for insulin-like growth factor 2 and ligands bearing mannose-6-phosphate, in particular lysosomal enzymes. We also show that this unique lipid is a specific antigen for human antibodies associated with the antiphospholipid syndrome. These antibodies may act intracellularly by altering the protein-sorting functions of endosomes.

Animals↗
PubMed1998

Lipids, lipid domains and lipid-protein interactions in endocytic membrane traffic.

It has long been known that lipids are responsible for the structural integrity of biological membranes, and confer specific dynamic properties to the bilayer. However, they have often been considered as passive components of the membrane, beyond their well-known function as second messengers in signal transduction pathways. Evidence has been accumulating that lipids are sorted along the routes of membrane traffic, and that they are directly involved in the regulation of protein sorting and membrane flow. In this review, we will discuss recent studies on the organization and possible functions of lipids in the endocytic pathway.

ADP-Ribosylation Factors↗
PubMed1997

Involvement of the transmembrane protein p23 in biosynthetic protein transport.

Here, we report the localization and characterization of BHKp23, a member of the p24 family of transmembrane proteins, in mammalian cells. We find that p23 is a major component of tubulovesicular membranes at the cis side of the Golgi complex (estimated density: 12,500 copies/micron2 membrane surface area, or approximately 30% of the total protein). Our data indicate that BHKp23-containing membranes are part of the cis-Golgi network/intermediate compartment. Using the G protein of vesicular stomatitis virus as a transmembrane cargo molecule, we find that p23 membranes are an obligatory station in forward biosynthetic membrane transport, but that p23 itself is absent from transport vesicles that carry the G protein to and beyond the Golgi complex. Our data show that p23 is not present to any significant extent in coat protein (COP) I-coated vesicles generated in vitro and does not colocalize with COP I buds and vesicles. Moreover, we find that p23 cytoplasmic domain is not involved in COP I membrane recruitment. Our data demonstrate that microinjected antibodies against the cytoplasmic tail of p23 inhibit G protein transport from the cis-Golgi network/ intermediate compartment to the cell surface, suggesting that p23 function is required for the transport of transmembrane cargo molecules. These observations together with the fact that p23 is a highly abundant component in the intermediate compartment, lead us to propose that p23 contributes to membrane structure, and that this contribution is necessary for efficient segregation and transport.

Amino Acid Sequence↗
PubMed1997

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.

Animals↗
PubMed1997

Generation of proteoliposomes from subcellular fractions.

Intracellular membranes are highly dynamic, yet they retain their identity and functional characteristics. Integral membrane proteins, which must confer this specific membrane identity, remain poorly characterized at the biochemical level, largely because detergent-mediated solubilization is required for purification and analysis, and several properties of integral membrane proteins can only be investigated when the molecule is properly embedded in a lipid bilayer. We present a method for the efficient reconstitution into proteoliposomes of integral membrane proteins from subcellular fractions. Integral membrane proteins were identified on high-resolution two-dimensional gels after selective extraction of soluble and peripheral membrane proteins; they accounted for 8% of the number of resolved polypeptides. A reconstitution procedure based on membrane solubilization with dodecyl-octaoxyethylene (C12E8) and subsequent detergent removal with BioBeads SM-2 resulted in the efficient reconstitution of several membrane proteins into proteoliposomes of uniform density. The generated proteoliposomes strongly resemble the starting membrane fraction in protein composition. This reconstitution allows the functional characterization of integral membrane proteins after enrichment and/or specific (immuno)depletion.

Animals↗
PubMed1997

Specific release of membrane-bound annexin II and cortical cytoskeletal elements by sequestration of membrane cholesterol.

Annexin II is an abundant protein which is present in the cytosol and on the cytoplasmic face of plasma membrane and early endosomes. It is generally believed that this association occurs via Ca(2+)-dependent binding to lipids, a mechanism typical for the annexin protein family. Although previous studies have shown that annexin II is involved in early endosome dynamics and organization, the precise biological role of the protein is unknown. In this study, we found that approximately 50% of the total cellular annexin was associated with membranes in a Ca(2+)-independent manner. This binding was extremely tight, since it resisted high salt and, to some extent, high pH treatments. We found, however, that membrane-associated annexin II could be quantitatively released by low concentrations of the cholesterol-sequestering agents filipin and digitonin. Both treatments released an identical and limited set of proteins but had no effects on other membrane-associated proteins. Among the released proteins, we identified, in addition to annexin II itself, the cortical cytoskeletal proteins alpha-actinin, ezrin and moesin, and membrane-associated actin. Our biochemical and immunological observations indicate that these proteins are part of a complex containing annexin II and that stability of the complex is sensitive to cholesterol sequestering agents. Since annexin II is tightly membrane-associated in a cholesterol-dependent manner, and since it seems to interact physically with elements of the cortical actin cytoskeleton, we propose that the protein serves as interface between membranes containing high amounts of cholesterol and the actin cytoskeleton.

Animals↗
PubMed1997

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.

Adenosine Triphosphate↗
PubMed1996

Endosomal fractions from viral K-ras-transformed MDCK cells reveal transformation specific changes on two-dimensional gel maps.

We have investigated the effects of viral Kirsten ras oncogene expression in Madin-Darby canine kidney (MDCK) II epithelial cell on the differential protein expression of organelle proteins. MDCK cells, stably transformed via infection with a helper-independent retroviral vector construct, were grown on permeable filter supports. Whereas normal cells form highly polarized monolayers, ras-transformed cells display an unpolarized phenotype, detaching from the substratum and developing multilayers (Schoenenberger, C.-A. et al., J. Cell Biol. 1991, 112, 873-889). We postulate that this breakdown of epithelial polarity reflects disturbed intracellular protein transport and sorting, namely, proteins will no longer be sorted correctly in intracellular organelles and will therefore not reach their appropriate target membrane. Here we emphasize the role of endosomes as sorting platform in epithelial cells. We found significant differences in the molecular composition of endosomes from normal vs. oncogenic transformed epithelial cells, strengthening previous evidence indicating that oncogenic transformation results in abnormal expression of normal genes (Celis, J. E., Olsen, E., Electrophoresis 1994, 15, 309-344) as well as the expression of new ones (Huber, L. A. et al., Electrophoresis 1994, 15, 468-473).

Animals↗
PubMed1996

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.

Animals↗
PubMed1995

Membrane transport in the endocytic pathway.

Despite controversies and debates, some fundamental properties of endosomes become apparent when comparing results from in vivo and in vitro strategies used to study endosomal membrane traffic. In addition, recent studies are starting to unravel the complex organization of early endosomes, in particular along the route followed by recycling receptors.

Animals↗