In B-lymphocytes, endocytosis of MHC I and MHC II molecules is important for the cross-priming and presentation of labile antigens, respectively. Here, we report that MHC I and MHC II were ...internalized by separate endocytic carriers that lacked transferrin receptor. Cholera toxin B was co-internalized with MHC II, but not with MHC I, suggesting that the CLIC/GEEC pathway is involved in the uptake of MHC II. Endocytosis of MHC I and MHC II was inhibited by filipin, but only MHC II showed a strong preference for a membrane raft environment in a co-clustering analysis with GM1. By using a novel method for the extraction of detergent-resistant membranes (DRMs), we observed that MHC I and MHC II associate with two distinct types of DRMs. These differ in density, protein content, lipid composition, and ultrastructure. The results of cell surface biotinylation and subsequent DRM isolation show that precursors for both DRMs coexist in the plasma membrane. Moreover, clustering of MHC proteins at the cell surface resulted in shifts of the respective DRMs, revealing proximity-induced changes in the membrane environment. Our results suggest that the preference of MHC I and MHC II for distinct membrane rafts directs them to different cellular entry points.
Epsin and AP180/CALM are important endocytic accessory proteins that are believed to be involved in the formation of clathrin coats. Both proteins associate with phosphorylated membrane inositol ...lipids through their epsin N-terminal homology domains and with other components of the endocytic machinery through short peptide motifs in their carboxyl-terminal segments. Using hydrodynamic and spectroscopic methods, we demonstrate that the parts of epsin 1 and AP180 that are involved in protein-protein interactions behave as poorly structured flexible polypeptide chains with little or no conventional secondary structure. The predominant cytosolic forms of both proteins are monomers. Furthermore, we show that recombinant epsin 1, like AP180, drives in vitro assembly of clathrin cages. We conclude that the epsin N-terminal homology domain-containing proteins AP180/CALM and epsin 1 have a very similar molecular architecture that is designed for the rapid and efficient recruitment of the principal coat components clathrin and AP-2 at the sites of coated pit assembly.
Rafting trips into the cell Lindner, Robert; Knorr, Ruth
Communicative & integrative biology,
20/9/1/, Letnik:
2, Številka:
5
Journal Article
Recenzirano
Odprti dostop
Lipid rafts are small, heterogeneous and short-lived assemblies of cholesterol, sphingolipids and few proteins in biological membranes. They can be converted to larger and more permanent membrane ...domains by coalescence. Cells appear to be able to modulate the size and the longevity of lipid rafts and thus exploit the local enrichment of membrane components for processes ranging from signaling to intracellular sorting and transport. In a recent paper, we provided evidence for the internalization of MHC I and MHC II along two distinct endocytosis pathways in mouse B-lymphocytes. Both pathways were much more dependent on membrane cholesterol than the clathrin-mediated uptake of transferrin receptor, which implicated lipid rafts in the internalization of MHC molecules. Indeed, MHC I and MHC II prefer distinct raft-like membrane environments as revealed by a co-clustering analysis with the sphingolipids GM1 and GM2. Moreover, MHC I and MHC II distributed to different types of detergent resistant membranes (DRMs) prepared by a novel detergent extraction procedure. In this article addendum we discuss the relationship between DRMs, small lipid rafts and stabilized rafts/membrane domains and propose a role for membrane domains in the endocytosis of MHC proteins.
Major histocompatibility complex class II protein (MHC II) molecules present antigenic peptides to CD4-positive T-cells. Efficient
T cell stimulation requires association of MHC II with membrane ...microdomains organized by cholesterol and glycosphingolipids
or by tetraspanins. Using detergent extraction at 37 °C combined with a modified flotation assay, we investigated the sequence
of events leading to the association of MHC II with cholesterol- and glycosphingolipid-rich membranes (DRMs) that are distinct
from tetraspanins. We find two stages of association of MHC II with DRMs. In stage one, complexes of MHC II and invariant
chain, a chaperone involved in MHC II transport, enter DRMs in the Golgi stack. In early endosomes, these complexes are almost
quantitatively associated with DRMs. Upon transport to late endocytic compartments, MHC II-bound invariant chain is stepwise
proteolyzed to the MHC class II-associated invariant chain peptide (CLIP) that remains MHC II-bound and retains a preference
for DRMs. At the transition between the two stages, CLIP is exchanged against processed antigens, and the resulting MHC II-peptide
complexes are transported to the cell surface. In the second stage, MHC II shows a lower overall association with DRMs. However,
surface MHC II molecules occupied with peptides that induce resistance to denaturation by SDS are enriched in DRMs relative
to SDS-sensitive MHC II-peptide complexes. Likewise, MHC II molecules loaded with long-lived processing products of hen-egg
lysozyme containing the immunodominant epitope 48â61 show a very high preference for DRMs. Thus after an initial mainly intracellular
stage of high DRM association, MHC II moves to a second stage in which its preference for DRMs is modulated by bound peptides.
In the metabolism of
Lactobacillus sanfranciscensis, the acetate kinase (AK) is a key enzyme and responsible for dephosphorylation of acetyl phosphate with the concomitant production of acetate and ...ATP. The
L. sanfranciscensis ack gene was identified by PCR methods. It encodes a 397 amino acid protein sharing 56% similarity with
Bacillus subtilis AK. Whereas cotranscription of
ack and
pta (phosphotransacetylase) is reported in previously characterised organisms, the
L. sanfranciscensis ack gene is not located in direct neighbourhood to the encoding gene. AK was heterologously expressed in
E. coli and characterised by its ν
max and K
m values and by the dependence of enzyme activity on temperature and pH. Based on this data the
in vivo role of the enzyme is discussed.
Rafting trips into the cell Lindner, Robert; Knorr, Ruth
Communicative & integrative biology,
2009, Letnik:
2, Številka:
5
Journal Article
Recenzirano
Odprti dostop
Lipid rafts are small, heterogeneous and short-lived assemblies of cholesterol, sphingolipids and few proteins in biological membranes. They can be converted to larger and more permanent membrane ...domains by coalescence. Cells appear to be able to modulate the size and the longevity of lipid rafts and thus exploit the local enrichment of membrane components for processes ranging from signaling to intracellular sorting and transport. In a recent paper, we provided evidence for the internalization of MHC I and MHC II along two distinct endocytosis pathways in mouse B-lymphocytes. Both pathways were much more dependent on membrane cholesterol than the clathrin-mediated uptake of transferrin receptor, which implicated lipid rafts in the internalization of MHC molecules. Indeed, MHC I and MHC II prefer distinct raft-like membrane environments as revealed by a co-clustering analysis with the sphingolipids G(M)1 and G(M)2. Moreover, MHC I and MHC II distributed to different types of detergent resistant membranes (DRMs) prepared by a novel detergent extraction procedure. In this article addendum we discuss the relationship between DRMs, small lipid rafts and stabilized rafts/membrane domains and propose a role for membrane domains in the endocytosis of MHC proteins.
Sourdough is the foremost cereal fermentation performed in a variety of technologies with almost any cereal. The lactobacilli studied most intensely include Lactobacillus sanfranciscensis, L. reuteri ...and L. pontis isolated from traditional and modern rye and wheat fermentations. Molecular biology tools are available for their rapid identification and monitoring throughout a process. The currently available insight on their metabolism can be used to explain their prevalence in this environment and their interactions. Key genes of the sugar degradation pathway were cloned and characterised from L. sanfranciscensis. In addition some strains were found to have special properties including the production of antagonistic compounds or the adhesion to human intestinal cells.
Intercellular adhesion molecule-1 (ICAM-1, CD54) is a ligand for the integrins lymphocyte function associated-1 (LFA-1, CD11a/CD18) and complement receptor-3 (Mac-1, CD11b/CD18) making it an ...important participant in many immune and inflammatory processes. Modified recombinant soluble ICAM-1 formed dimers. This result indicated that the ectodomain of ICAM-1 contains homophilic interaction sites. Soluble ICAM-1 dimers bind to solid-phase purified LFA-1 with high avidity (dissociation constant Kd = 8 nM) in contrast to soluble ICAM-1 monomers whose binding was not measurable. Cell surface ICAM-1 was found to be dimeric based on two distinct criteria. First, a monoclonal antibody specific for monomeric soluble ICAM-1, CA7, binds normal ICAM-1 poorly at the cell surface; this antibody, however, binds strongly to two mutant forms of ICAM-1 when expressed at the cell surface, thus identifying elements required for dimer formation. Second, chemical cross-linking of cell surface ICAM-1 on transfected cells and tumor necrosis factor-activated endothelial cells results in conversion of a portion of ICAM-1 to a covalent dimer. Cell surface ICAM-1 dimers are more potent ligands for LFA-1-dependent adhesion than ICAM-1 monomers. While many extracellular matrix-associated ligands of integrins are multimeric, this is the first evidence of specific, functionally important homodimerization of a cell surface integrin ligand.
The I domain of lymphocyte function-associated antigen (LFA)-1 contains an intercellular adhesion molecule (ICAM)-1 and ICAM-3 binding site, but the relationship of this site to regulated adhesion is ...unknown. To study the adhesive properties of the LFA-1 I domain, we stably expressed a GPI-anchored form of this I domain (I-GPI) on the surface of baby hamster kidney cells. I-GPI cells bound soluble ICAM-1 (sICAM-1) with a low avidity and affinity. Flow cell experiments demonstrated a specific rolling interaction of I-GPI cells on bilayers containing purified full length ICAM-1 or ICAM-3. The LFA-1 activating antibody MEM-83, or its Fab fragment, decreased the rolling velocity of I-GPI cells on ICAM-1-containing membranes. In contrast, the interaction of I-GPI cells with ICAM-3 was blocked by MEM-83. Rolling of I-GPI cells was dependent on the presence of Mg2+. Mn2+ only partially substituted for Mg2+, giving rise to a small fraction of rolling cells and increased rolling velocity. This suggests that the I domain acts as a transient, Mg2+-dependent binding module that cooperates with another Mn2+-stimulated site in LFA-1 to give rise to the stable interaction of intact LFA-1 with ICAM-1.
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