scholarly journals A dileucine signal situated in the C-terminal tail of the lysosomal membrane protein p40 is responsible for its targeting to lysosomes

2008 ◽  
Vol 414 (3) ◽  
pp. 431-440 ◽  
Author(s):  
Marielle Boonen ◽  
Roberta Rezende de Castro ◽  
Gaëlle Cuvelier ◽  
Isabelle Hamer ◽  
Michel Jadot
Keyword(s):  
Subcellular Fractionation ◽  
Lysosomal Membrane ◽  
Consensus Sequences ◽  
Cell Surface Biotinylation ◽  
Dileucine Motif ◽  
Lysosomal Sorting ◽  
Critical Residues ◽  
Lysosomal Membrane Proteins ◽  
Golgi Network

Transport of newly synthesized lysosomal membrane proteins from the TGN (trans-Golgi network) to the lysosomes is due to the presence of specific signals in their cytoplasmic domains that are recognized by cytosolic adaptors. p40, a hypothetical transporter of 372 amino acids localized in the lysosomal membrane, contains four putative lysosomal sorting motifs in its sequence: three of the YXXϕ-type (Y6QLF, Y106VAL, Y333NGL) and one of the [D/E]XXXL[L/I]-type (EQERL360L361). To test the role of these motifs in the biosynthetic transport of p40, we replaced the most critical residues of these consensus sequences, the tyrosine residue or the leucine–leucine pair, by alanine or alanine–valine respectively. We analysed the subcellular localization of the mutated p40 proteins in transfected HeLa cells by confocal microscopy and by biochemical approaches (subcellular fractionation on self-forming Percoll density gradients and cell surface biotinylation). The results of the present study show that p40 is mistargeted to the plasma membrane when its dileucine motif is disrupted. No role of the tyrosine motifs could be put forward. Taken together, our results provide evidence that the sorting of p40 from the TGN to the lysosomes is directed by the dileucine EQERL360L361 motif situated in its C-terminal tail.

scholarly journals Antibodies to platelets in patients with anti-phospholipid antibodies

Blood ◽  
1991 ◽  
Vol 77 (12) ◽  
pp. 2655-2659 ◽  
Author(s):  
HJ Out ◽  
PG de Groot ◽  
M van Vliet ◽  
GC de Gast ◽  
HK Nieuwenhuis ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Lysosomal Membrane ◽  
Immunofluorescence Method ◽  
Exact Role ◽  
Flow Cytofluorometry ◽  
Healthy Donors ◽  
Lysosomal Membrane Proteins ◽  
Lysosomal Membrane Protein ◽  
Platelet Autoantibodies

Abstract Binding of anti-phospholipid antibodies to circulating platelets and its consequences on platelet activation and aggregation was investigated in 11 patients with anti-phospholipid antibodies. Seven patients had mild thrombocytopenia. Nine healthy donors served as controls. Binding to platelets was investigated by performing enzyme- linked immunosorbent assays (ELISAs) with phospholipids as antigen on platelet eluates. Platelet activation was measured by flow cytofluorometry using monoclonal antibodies to an activation-specific lysosomal membrane protein. Findings in ELISA were compared with results of a conventional immunofluorescence method to detect platelet autoantibodies. In seven patients antibodies to negatively charged phospholipids were present in platelet eluates. In all thrombocytopenic patients and controls the platelets were not activated and aggregation was not impaired. There was a positive concordance of 50% between the results of immunofluorescence and ELISA. No apparent relation was found between the results of ELISA or immunofluorescence and platelet counts. It is concluded that anti-phospholipid antibodies can bind to circulating platelets. This binding is not associated with measurable aggregation abnormalities nor with platelet activation characterized by exposure of lysosomal membrane proteins. More studies are necessary to determine the exact role of anti-phospholipid antibodies in the pathogenesis of thrombocytopenia and thrombosis.

scholarly journals Intracellular localization of p40, a protein identified in a preparation of lysosomal membranes

2006 ◽  
Vol 395 (1) ◽  
pp. 39-47 ◽  
Author(s):  
Marielle Boonen ◽  
Isabelle Hamer ◽  
Muriel Boussac ◽  
Anne-Françoise Delsaute ◽  
Bruno Flamion ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Intracellular Localization ◽  
Subcellular Fractionation ◽  
Lysosomal Membrane ◽  
Knowing That ◽  
Lysosomal Membrane Proteins ◽  
Green Fluorescent ◽  
Rat Liver Lysosomes ◽  
Lysosome Membrane

Unlike lysosomal soluble proteins, few lysosomal membrane proteins have been identified. Rat liver lysosomes were purified by centrifugation on a Nycodenz density gradient. The most hydrophobic proteins were extracted from the lysosome membrane preparation and were identified by MS. We focused our attention on a protein of approx. 40 kDa, p40, which contains seven to ten putative transmembrane domains and four lysosomal consensus sorting motifs in its sequence. Knowing that preparations of lysosomes obtained by centrifugation always contain contaminant membranes, we combined biochemical and morphological methods to analyse the subcellular localization of p40. The results of subcellular fractionation of mouse liver homogenates validate the lysosomal residence of p40. In particular, a density shift of lysosomes induced by Triton WR-1339 similarly affected the distributions of p40 and β-galactosidase, a lysosomal marker protein. We confirmed by fluorescence microscopy on eukaryotic cells transfected with p40 or p40–GFP (green fluorescent protein) constructs that p40 is localized in lysosomes. A first molecular characterization of p40 in transfected Cos-7 cells revealed that it is an unglycosylated protein tightly associated with membranes. Taken together, our results strongly support the hypothesis that p40 is an authentic lysosomal membrane protein.

Lysosomal membrane proteins: life between acid and neutral conditions

2010 ◽  
Vol 38 (6) ◽  
pp. 1420-1423 ◽  
Author(s):  
Paul Saftig ◽  
Bernd Schröder ◽  
Judith Blanz
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Hydrolytic Degradation ◽  
Cell Types ◽  
Transport Processes ◽  
Lysosomal Membrane ◽  
Cell Physiology ◽  
Depth Analysis ◽  
Lysosomal Membrane Proteins

Whereas we have a profound understanding about the function and biogenesis of the protein constituents in the lumen of the lysosomal compartment, much less is known about the functions of proteins of the lysosomal membrane. Proteomic analyses of the lysosomal membrane suggest that, apart from the well-known lysosomal membrane proteins, additional and less abundant membrane proteins are present. The identification of disease-causing genes and the in-depth analysis of knockout mice leading to mutated or absent membrane proteins of the lysosomal membrane have demonstrated the essential role of these proteins in lysosomal acidification, transport of metabolites resulting from hydrolytic degradation and interaction and fusion with other cellular membrane systems. In addition, trafficking pathways of lysosomal membrane proteins are closely linked to the biogenesis of this compartment. This is exemplified by the recent finding that LIMP-2 (lysosomal integral membrane protein type-2) is responsible for the mannose 6-phosphate receptor-independent delivery of newly synthesized β-glucocerebrosidase to the lysosome. Similar to LIMP-2, which could also be linked to vesicular transport processes in certain polarized cell types, the major constituents of the lysosomal membrane, the glycoproteins LAMP (lysosome-associated membrane protein)-1 and LAMP-2 are essential for regulation of lysosomal motility and participating in control of membrane fusion events between autophagosomes or phagosomes with late endosomes/lysosomes. Our recent investigations into the role of these proteins have not only increased our understanding of the endolysosomal system, but also supported their major role in cell physiology and the development of different diseases.

scholarly journals Antibodies to platelets in patients with anti-phospholipid antibodies

Blood ◽  
1991 ◽  
Vol 77 (12) ◽  
pp. 2655-2659 ◽  
Author(s):  
HJ Out ◽  
PG de Groot ◽  
M van Vliet ◽  
GC de Gast ◽  
HK Nieuwenhuis ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Lysosomal Membrane ◽  
Immunofluorescence Method ◽  
Exact Role ◽  
Flow Cytofluorometry ◽  
Healthy Donors ◽  
Lysosomal Membrane Proteins ◽  
Lysosomal Membrane Protein ◽  
Platelet Autoantibodies

Binding of anti-phospholipid antibodies to circulating platelets and its consequences on platelet activation and aggregation was investigated in 11 patients with anti-phospholipid antibodies. Seven patients had mild thrombocytopenia. Nine healthy donors served as controls. Binding to platelets was investigated by performing enzyme- linked immunosorbent assays (ELISAs) with phospholipids as antigen on platelet eluates. Platelet activation was measured by flow cytofluorometry using monoclonal antibodies to an activation-specific lysosomal membrane protein. Findings in ELISA were compared with results of a conventional immunofluorescence method to detect platelet autoantibodies. In seven patients antibodies to negatively charged phospholipids were present in platelet eluates. In all thrombocytopenic patients and controls the platelets were not activated and aggregation was not impaired. There was a positive concordance of 50% between the results of immunofluorescence and ELISA. No apparent relation was found between the results of ELISA or immunofluorescence and platelet counts. It is concluded that anti-phospholipid antibodies can bind to circulating platelets. This binding is not associated with measurable aggregation abnormalities nor with platelet activation characterized by exposure of lysosomal membrane proteins. More studies are necessary to determine the exact role of anti-phospholipid antibodies in the pathogenesis of thrombocytopenia and thrombosis.

scholarly journals The role of lysosomal membrane proteins in glucose and lipid metabolism

2021 ◽  
Vol 35 (10) ◽  
Author(s):  
Jing Gu ◽  
Mengya Geng ◽  
Mengxiang Qi ◽  
Lizhuo Wang ◽  
Yao Zhang ◽  
...  
Keyword(s):  
Lipid Metabolism ◽  
Membrane Proteins ◽  
Lysosomal Membrane ◽  
Glucose And Lipid Metabolism ◽  
Lysosomal Membrane Proteins

On the Role of Transferrin in 67Ga Uptake by Tumor Cells

1988 ◽  
Vol 27 (04) ◽  
pp. 151-153
Author(s):  
P. Thouvenot ◽  
F. Brunotte ◽  
J. Robert ◽  
L. J. Anghileri
Keyword(s):  
Specific Binding ◽  
Subcellular Fractionation ◽  
Animal Blood ◽  
Tumor Bearing ◽  
Cell Structures ◽  
The Difference ◽  
Sarcoma Cells ◽  
In Vitro Uptake

In vitro uptake of 67Ga-citrate and 59Fe-citrate by DS sarcoma cells in the presence of tumor-bearing animal blood plasma showed a dramatic inhibition of both 67Ga and 59Fe uptakes: about ii/io of 67Ga and 1/5o of the 59Fe are taken up by the cells. Subcellular fractionation appears to indicate no specific binding to cell structures, and the difference of binding seems to be related to the transferrin chelation and transmembrane transport differences

Lysosomal membrane proteins exposed to melanin in melanocyte B16F10

2016 ◽  
Vol 8 (3) ◽  
pp. 253-257
Author(s):  
Seung Hyuck Bang ◽  
Dong Jun Park ◽  
Yang-Hoon Kim ◽  
Jiho Min
Keyword(s):  
Membrane Proteins ◽  
Lysosomal Membrane ◽  
Lysosomal Membrane Proteins

scholarly journals Sorting of mannose 6-phosphate receptors and lysosomal membrane proteins in endocytic vesicles.

1988 ◽  
Vol 107 (6) ◽  
pp. 2491-2501 ◽  
Author(s):  
H J Geuze ◽  
W Stoorvogel ◽  
G J Strous ◽  
J W Slot ◽  
J E Bleekemolen ◽  
...  
Keyword(s):  
Lysosomal Membrane ◽  
Membrane Glycoprotein ◽  
Horse Radish Peroxidase ◽  
Western Blots ◽  
Lysosomal Membrane Proteins ◽  
Mannose 6 Phosphate ◽  
Lysosomal Membrane Glycoprotein ◽  
Endocytic Vesicles ◽  
Kinetics Of ◽  
Combined Data

The intracellular distributions of the cation-independent mannose 6-phosphate receptor (MPR) and a 120-kD lysosomal membrane glycoprotein (lgp120) were studied in rat hepatoma cells. Using quantitative immunogold cytochemistry we found 10% of the cell's MPR located at the cell surface. In contrast, lgp120 was not detectable at the plasma membrane. Intracellularly, MPR mainly occurred in the trans-Golgi reticulum (TGR) and endosomes. lgp120, on the other hand, was confined to endosomes and lysosomes. MPR was present in both endosomal tubules and vacuoles, whereas lgp120 was confined to the endosomal vacuoles. In cells incubated for 5-60 min with the endocytic tracer cationized ferritin, four categories of endocytic vacuoles could be discerned, i.e., vacuoles designated MPR+/lgp120-, MPR+/lgp120+, MPR-/lgp120+, and vacuoles nonimmunolabeled for MPR and lgp120. Tracer first reached MPR+/lgp120-, then MPR+/lgp120+, and finally MPR-/lgp120+ vacuoles, which are assumed to represent lysosomes. To study the kinetics of appearance of endocytic tracers in MPR-and/or lgp120-containing pools in greater detail, cells were allowed to endocytose horse-radish peroxidase (HRP) for 5-90 min. The reduction in detectability of MPR and lgp120 antigenicity on Western blots, due to treatment of cell homogenates with 3'3-diaminobenzidine, was followed in time. We found that HRP reached the entire accessible pool of MPR almost immediately after internalization of the tracer, while prolonged periods of time were required for HRP to maximally access lgp120. The combined data suggest that MPR+/lgp120+ vacuoles are endocytic vacuoles, intermediate between MPR+/lgp120-endosomes and MPR-/lgp120+ lysosomes, and represent the site where MPR is sorted from lgp120 destined for lysosomes. We propose that MPR is sorted from lgp120 by selective lateral distribution of the receptor into the tubules of this compartment, resulting in the retention of lgp120 in the vacuoles and the net transport of lgp120 to lysosomes.

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