Bjørn Bremnes

FYVE fingers bind PtdIns(3)P [5]

The membrane lipid phosphatidylinositol-3-phosphate (PtdIns(3)P) is constitutively produced by yeast and higher eukaryotes through the phosphorylation of phosphatidylinositol by phosphatidylinositol-3-OH kinase (PI(3)K). PtdIns(3)P is important for vesicular transport, but little is known about how it acts, and proteins that specifically recognize it have not yet been identified. Here we identify the FYVE finger, an evolutionarily conserved double-zinc-binding domain (see Supplementary information), as a protein structure that binds to PtdIns(3)P with high specificity.

Syntaxin-16, a putative Golgi t-SNARE

Members of the syntaxin family of integral membrane proteins have recently been implicated as vesicle receptors on target membranes, coresponsible for the specificity of intracellular membrane traffic. So far, only a small number of different mammalian syntaxins have been identified. Here we report the cloning of three new human syntaxin cDNAs, presumably originating from alternative splicing of the same transcript. Syntaxin-16A and syntaxin-16B are identical, except that the latter contains an insertion of 21 amino acid residues. Syntaxin-16C is a truncated version of syntaxin-16A, lacking the C-terminal coiled-coil and hydrophobic regions characteristic for syntaxins. Database searches identified putative yeast, plant and nematode homologues of syntaxin-16, indicating that this protein is conserved through evolution, and syntaxin-16 belongs to a new subgroup of syntaxins. Epitope-tagged syntaxin-16A and syntaxin-16B were found to colocalize with the Golgi marker beta-COP, while syntaxin-16C was found in the cytosol. Syntaxin-16A associates posttranslationally with microsomes, and appears to be transported to the Golgi via the endoplasmic reticulum. The three syntaxin-16 forms may have differential roles in intracellular trafficking.

Structure-Activity Relationship of the Leucine-based Sorting Motifs in the Cytosolic Tail of the Major Histocompatibility Complex-associated Invariant Chain

The cytosolic tail of the major histocompatibility complex-associated invariant chain protein contains two Leu-based motifs that both mediate efficient sorting to the endocytic pathway. Nuclear magnetic resonance data on a peptide of 27 residues corresponding to the cytosolic tail of human invariant chain indicate that in water at pH 7.4 the membrane distal motif Leu7-Ile8 lies within a nascent helix, while the membrane proximal motif Met16-Leu17 is part of a turn. The presence of a small amount of methanol stabilizes an α helix from Gln4 to Leu17 with a kink on Pro15. Point mutations of the cytosolic tail of the protein suggest that amino-terminal residues located in spatial proximity to the Leu motifs contribute to efficient internalization and targeting to endosomes in transfected COS cells. Residues on the spatially opposite side of the Leu motifs were, on the other hand, mutated with no measurable effect on targeting. Structural and biological data thus suggest that the signals are not continuous but consist of “signal patches” formed by the three-dimensional structure of the cytosolic tail of invariant chain.

Human IgG3 can adopt the disulfide bond pattern characteristic for IgG1 without resembling it in complement mediated cell lysis

In this paper we describe the construction of mouse-human IgG3 mutant antibodies resembling IgG1 in their disulfide bond pattern between the heavy and light chain (H-L) and between the two heavy chains (H-H). The effector functions of these mutant antibodies were compared to normal IgG3 and IgG1. Changing only the disulfide bond pattern between the heavy and light chains did not alter the ability to induce complement mediated cell lysis (CML), regardless of the amount of corresponding antigen that had been introduced to the surface of the target cells. However, alteration of the disulfide bond pattern between the two heavy chains had a large effect on CML due to shortening of the hinge from 62 to 15 amino acids. No difference between the mutants and normal antibodies in antibody-dependent cell-mediated cytotoxicity (ADCC) was observed. This suggests that IgG3 can adopt the H-L disulfide bond pattern of IgG1 without obtaining the CML activity characteristic for IgG1.

FYVE finger proteins as effectors of phosphatidylinositol 3-phosphate.

Phosphatidylinositol 3-phosphate (PtdIns(3)P), generated via the phosphorylation of phosphatidylinositol by phosphatidylinositol 3-kinase (PI 3-kinase), plays an essential role in intracellular membrane traffic. The underlying mechanism is still not understood in detail, but the recent identification of the FYVE finger as a protein domain that binds specifically to PtdIns(3)P provides a number of potential effectors for PtdIns(3)P. The FYVE finger (named after the first letter of the four proteins containing it; Fab1p, YOTB, Vac1p and EEA1) is a double-zinc binding domain that is conserved in more than 30 proteins from yeast to mammals. It is found in several proteins involved in intracellular traffic, and FYVE finger mutations that affect zinc binding are associated with the loss of function of several of these proteins. The interaction of FYVE fingers with PtdIns(3)P may serve three alternative functions: First, to recruit cytosolic FYVE finger proteins to PtdIns(3)P-containing membranes (in concert with accessory molecules); second, to enrich for membrane bound FYVE finger proteins into PtdIns(3)P containing microdomains within the membrane; and third, to modulate the activity of membrane bound FYVE finger proteins.

The leucine-based motif DDQxxLI is recognized both for internalization and basolateral sorting of invariant chain in MDCK cells.

The major histocompatibility complex (MHC) class II-associated invariant chain (Ii) contains signals for transport to endocytic compartments where the class II molecules bind antigenic peptides for presentation to CD4+ T cells. Two leucine-based signals in the Ii cytoplasmic tail can be independently recognized for endosomal sorting of Ii, and we have recently shown that each signal is sufficient for basolateral sorting and internalization of Ii in polarized Madine Darby Canine Kidney (MDCK) II cells. The recognition motif for endosomal sorting is complex and consists of two critical leucine-like residues as well as surrounding amino acids. Here, we have analyzed the importance of residues surrounding the membrane-distal leucine-based signal in basolateral sorting and internalization of Ii in MDCK II cells. We find that the DDQxxLI motif is involved in both sorting events indicating the presence of similar signal recognition components both at the TGN and at the plasma membrane. The identical motif is required for endosomal localization and internalization of Ii also in simian COS cells and the human HeLa and M1 cells.

A new triple-stranded α-helical bundle in solution: the assembling of the cytosolic tail of MHC-associated invariant chain

BACKGROUND The invariant chain (li) is a transmembrane protein that associates with the major histocompatibility complex class II (MHC II) molecules in the endoplasmic reticulum. The cytosolic tail of li contains two leucine-based sorting motifs and is involved in sorting the MHC II molecules to the endosomal pathway where the peptide antigen is bound. This region of li also contributes to phenotypical changes in cells, such as the formation of large endocytic structures. RESULTS We report here the three-dimensional structure of a 27 amino acid peptide corresponding to the cytosolic tail of li. The structure was determined by nuclear magnetic resonance (NMR) spectroscopy using a computational strategy. At high concentration, this structure reveals a new triple-stranded alpha-helical bundle in which the helices, two parallel and one antiparallel, are almost coplanar. Trimerization is mediated by electrostatic interactions intercalated by three hydrophobic layers. CONCLUSIONS The new trimer fold, the first to be identified by NMR data alone, can be used to improve understanding of protein-protein interactions and to model multiple-helical transmembrane proteins and receptors. We suggest that interactions of the li cytosolic tails may form part of a mechanism that could cause the endosomal retention and enlarged endosomes induced by li.