Martin Sachse

University of Liverpool, Liverpool, England, United Kingdom

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Publications (8)38.96 Total impact

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    ABSTRACT: Epidermal growth factor receptors (EGFRs) destined for lysosomal degradation are sorted in the early endosomal vacuole into small, lumenal vesicles that arise by inward budding of the limiting membrane. We have previously shown that, before their incorporation into internal vesicles, EGFRs are concentrated in flat bilayered-clathrin coats on the endosomal vacuole. Here, we show that an ATPase-deficient mutant of hVPS4 (hVPS4(EQ)) increases the association of bilayered coats with endosomal vacuoles. In addition, hVPS4(EQ) leads to a reduction in the number of internal vesicles in early and late endosomal vacuoles, and retention of EGFRs at the limiting membrane. Interestingly, hVPS4(EQ) was predominantly found on non-coated regions of endosomal vacuoles, often at the rim of a coated area. In line with published data on Vps4p function in yeast, these results suggest that hVPS4 is involved in the release of components of the bilayered coat from the endosomal membrane. Moreover, our data suggest that disassembly of the coat is required for the formation of internal vesicles.
    Journal of Cell Science 05/2004; 117(Pt 9):1699-708. · 5.88 Impact Factor
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    ABSTRACT: The growth hormone (GH) receptor is a key regulator of cellular metabolism. Unlike most growth factor receptors, its downregulation is not initiated by its ligand. Like many growth factor receptors, specific molecular mechanisms guarantee that a receptor can signal only once in its lifetime. Three features render the GH receptor unique: (a) an active ubiquitination system is required for both uptake (endocytosis) and degradation in the lysosomes; (b) uptake of the receptor is a continuous process, independent of both GH binding and Jak2 signal transduction; (c) only the cell surface expression of dimerised GH receptors is controlled by the ubiquitin system. This system enables two independent regulatory mechanisms for the endocrinology of the GH/GHR axis: the pulsatile secretion of GH by the pituitary and the GH sensitivity of individual cells of the body by the effects of the ubiquitin system on GH receptor availability.
    Current topics in microbiology and immunology 02/2004; 286:81-118. · 4.86 Impact Factor
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    ABSTRACT: Hepatocyte growth factor regulated tyrosine kinase substrate (Hrs), a main component of the 'bilayered' clathrin coat on sorting endosomes, was originally identified as a substrate of activated tyrosine kinase receptors. We have analysed Hrs phosphorylation in response to epidermal growth factor (EGF) stimulation and show that the evolutionary conserved tyrosines Y329 and Y334 provide the principal phosphorylation sites. Hrs is proposed to concentrate ubiquitinated receptors within clathrin-coated regions via direct interaction with its UIM (ubiquitin interaction motif) domain. We show that the same UIM domain is necessary for EGF-stimulated tyrosine phosphorylation of Hrs. Over-expression of wild-type Hrs or a double mutant, Y329/334F, defective in EGF-dependent phosphorylation, both substantially retard EGF receptor (EGFR) degradation by inhibiting internal vesicle formation and thereby preventing EGFR incorporation into lumenal vesicles of the multivesicular bodies. In contrast, mutation or deletion of the Hrs-UIM domain strongly suppresses this effect. In addition the UIM-deletion and point mutants are also observed on internal membranes, indicating a failure to dissociate from the endosomal membrane prior to incorporation of the receptor complex into lumenal vesicles. Our data suggest a role for the UIM-domain of Hrs in actively retaining EGFR at the limiting membrane of endosomes as a prelude to lumenal vesicle formation.
    Journal of Cell Science 11/2003; 116(Pt 20):4169-79. · 5.88 Impact Factor
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    ABSTRACT: In many cells endosomal vacuoles show clathrin coats of which the function is unknown. Herein, we show that this coat is predominantly present on early endosomes and has a characteristic bilayered appearance in the electron microscope. By immunoelectron microscopy we show that the coat contains clathrin heavy as well as light chain, but lacks the adaptor complexes AP1, AP2, and AP3, by which it differs from clathrin coats on endocytic vesicles and recycling endosomes. The coat is insensitive to short incubations with brefeldin A, but disappears in the presence of the phosphatidylinositol 3-kinase inhibitor wortmannin. No association of endosomal coated areas with tracks of tubulin or actin was found. By quantitative immunoelectron microscopy, we found that the lysosomal-targeted receptors for growth hormone (GHR) and epidermal growth factor are concentrated in the coated membrane areas, whereas the recycling transferrin receptor is not. In addition, we found that the proteasomal inhibitor MG 132 induces a redistribution of a truncated GHR (GHR-369) toward recycling vesicles, which coincided with a redistribution of endosomal vacuole-associated GHR-369 to the noncoated areas of the limiting membrane. Together, these data suggest a role for the bilayered clathrin coat on vacuolar endosomes in targeting of proteins to lysosomes.
    Molecular Biology of the Cell 05/2002; 13(4):1313-28. · 4.60 Impact Factor
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    ABSTRACT: In most cells the endocytic system is organized following a common concept that allows for the integrative handling of a variety of housekeeping functions. In addition, variations on the general scheme provide for specialized endosome-based pathways that occur only in specific cell types. The diversity of endosomal functions is not only reflected by characteristic molecular compositions, but also mirrored in their morphological organization. In this review we will first describe the general outline of the endocytic system by combining kinetic, morphological, molecular, and functional definitions. In the second part, adaptations of endosomes that allow their functioning in specialized processes, such as antigen presentation, synaptic vesicle formation, and glucose transport, will be addressed.
    Histochemie 03/2002; 117(2):91-104. · 2.61 Impact Factor
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    ABSTRACT: Endocytosis of the growth hormone receptor (GHR) requires an active ubiquitin-conjugation system. In addition, it depends on a 10 amino acid residues motif in the GHR-cytoplasmic tail, the ubiquitin dependent-endocytosis or UbE-motif. To gain insight into the role of ubiquitination in the early steps of endocytosis, we performed an ultrastructural analysis of GH-uptake in Chinese hamster cells expressing wild-type or mutant GHRs. In wild-type GHR cells, GH was found to be exclusively taken up via clathrin-coated pits. In early endosomes it was efficiently sorted from recycling transferrin and targeted to the degradative pathway. Mutation of all lysine residues of a truncated GHR (GHR-399K-) precludes ubiquitination of the receptor, but internalization of GHR-399K- still depends on an active ubiquitin system. We found that GHR-399K- incorporates GH into clathrin-coated vesicles with the same efficiency as wild-type GHR. By contrast, a mutation in the UbE-motif (GHR-F327A) largely abolished incorporation of GH into clathrin-coated vesicles. Notably, access of GH to clathrin-coated lattices was not affected in GHR-F327A cells. These data corroborate and extend previous data that the UbE-motif but not ubiquitination of the receptor itself recruits GHR into clathrin-coated vesicles. Moreover, they suggest that incorporation of GHR into clathrin-coated lattices is differentially regulated from incorporation into clathrin-coated pits.
    Journal of Cell Science 12/2001; 114(Pt 21):3943-52. · 5.88 Impact Factor
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    ABSTRACT: The ubiquitin-proteasome pathway acts as a regulator of the endocytosis of selected membrane proteins. Recent evidence suggests that it may also function in the intracellular trafficking of membrane proteins. In this study, several models were used to address the role of the ubiquitin-proteasome pathway in sorting of internalized proteins to the lysosome. We found that lysosomal degradation of ligands, which remain bound to their receptors within the endocytic pathway, is blocked in the presence of specific proteasome inhibitors. In contrast, a ligand that dissociates from its receptor upon endosome acidification is degraded under the same conditions. Quantitative electron microscopy showed that neither the uptake nor the overall distribution of the endocytic marker bovine serum albumin-gold is substantially altered in the presence of a proteasome inhibitor. The data suggest that the ubiquitin-proteasome pathway is involved in an endosomal sorting step of selected membrane proteins to lysosomes, thereby providing a mechanism for regulated degradation.
    Molecular Biology of the Cell 09/2001; · 4.60 Impact Factor
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    ABSTRACT: Endocytosis of the growth hormone receptor (GHR) depends on a functional ubiquitin conjugation system. A 10-amino acid residue motif within the GHR cytosolic tail (the ubiquitin-dependent endocytosis motif) is involved in both GHR ubiquitination and endocytosis. As shown previously, ubiquitination of the receptor itself is not required. In this paper ubiquitination of the GHR was used as a tool to address the question of at which stage the ubiquitin conjugation system acts in the process of GHR endocytosis. If potassium depletion was used to interfere with early stages of coated pit formation, both GHR endocytosis and ubiquitination were inhibited. Treatment of cells with methyl-beta-cyclodextrin inhibited endocytosis at the stage of coated vesicle formation. Growth hormone addition to methyl-beta-cyclodextrin-treated cells resulted in an accumulation of ubiquitinated GHR at the cell surface. Using immunoelectron microscopy, the GHR was localized in flattened clathrin-coated membranes. In addition, when clathrin-mediated endocytosis was inhibited in HeLa cells expressing a temperature-sensitive dynamin mutant, ubiquitinated GHR accumulated at the cell surface. Together, these data show that the GHR is ubiquitinated at the plasma membrane, before endocytosis occurs, and indicate that the resident time of the GHR at the cell surface is regulated by the ubiquitin conjugation system together with the endocytic machinery.
    Journal of Biological Chemistry 03/2001; 276(6):3778-84. · 4.65 Impact Factor