An acidic sequence within the cytoplasmic domain of furin functions as a determinant of trans-Golgi network localization and internalization from the cell surface.

The mammalian endopeptidase, furin, is predominantly localized to the trans-Golgi network (TGN) at steady state. The localization of furin to this compartment seems to be the result of a dynamic process in which the protein undergoes cycling between the TGN and the plasma membrane. Both TGN localization and internalization from the plasma membrane are mediated by targeting information contained within the cytoplasmic domain of furin.

A lysosomal targeting signal in the cytoplasmic tail of the beta chain directs HLA-DM to MHC class II compartments.

In human B cells, class II molecules of the major histocompatibility complex (MHC-II) accumulate in an endosomal/lysosomal compartment, the MIIC, in which they may encounter and bind peptides. An additional molecule required for MHC-II peptide binding, HLA-DM (DM), has also been localized to the MIIC. Neither the relationship of the MIIC to the endosomal system nor the mechanisms by which DM localizes to the MIIC are understood.

Major histocompatibility complex class II compartments in human B lymphoblastoid cells are distinct from early endosomes.

In human B lymphoblastoid cell lines, the majority of major histocompatibility complex (MHC) class II heterodimers are located on the cell surface and in endocytic compartments, while invariant chain (Ii)-associated class II molecules represent biosynthetic intermediates which are present mostly in the endoplasmic reticulum and Golgi complex. To investigate the origin of the MHC class II-positive compartments and their relation to early endosomes, the intracellular distribution of MHC class II molecules and Ii in relation to endocytic tracers was studied in human lymphoblastoid B cells by immunoelectronmicroscopy on ultrathin cryosections. Cross-linking of surface immunoglobulins, followed by a brief period of internalization of the immune complexes, did not alter the intracellular distribution of MHC class II molecules.

Overexpression of wild-type and mutant ARF1 and ARF6: distinct perturbations of nonoverlapping membrane compartments.

The ARF GTP binding proteins are believed to function as regulators of membrane traffic in the secretory pathway. While the ARF1 protein has been shown in vitro to mediate the membrane interaction of the cytosolic coat proteins coatomer (COP1) and gamma-adaptin with the Golgi complex, the functions of the other ARF proteins have not been defined. Here, we show by transient transfection with epitope-tagged ARFs, that whereas ARF1 is localized to the Golgi complex and can be shown to affect predictably the assembly of COP1 and gamma-adaptin with Golgi membranes in cells, ARF6 is localized to the endosomal/plasma membrane system and has no effect on these Golgi-associated coat proteins.

The cytoplasmic domain mediates localization of furin to the trans-Golgi network en route to the endosomal/lysosomal system.

To investigate the mechanisms of membrane protein localization to the Golgi complex, we have examined the intracellular trafficking of epitope-tagged forms of the mammalian endopeptidase, furin, in stably transformed rat basophilic leukemia cells. Our studies show that furin is predominantly localized to the trans-Golgi network (TGN) at steady state, with smaller amounts present in intracellular vesicles. Biochemical and morphological analyses reveal that furin is progressively delivered to a lysosomal compartment, where it is degraded.

Chemically Induced Cuticle Mutation Affecting Epidermal Conductance to Water Vapor and Disease Susceptibility in Sorghum bicolor (L.) Moench.

Analysis of Sorghum bicolor bloomless (bm) mutants with altered epicuticular wax (EW) structure uncovered a mutation affecting both EW and cuticle deposition. The cuticle of mutant bm-22 was about 60% thinner and approximately one-fifth the weight of the wild-type parent P954035 (WT-P954035) cuticles. Reduced cuticle deposition was associated with increased epidermal conductance to water vapor.

An activating mutation in ARF1 stabilizes coatomer binding to Golgi membranes.

The Ras-related protein ADP-ribosylation factor 1 (ARF1) is a low molecular weight GTP binding protein, which in its GTP state supports the binding of coatomer, a cytosolic coat protein complex, to Golgi membranes. To create an "active" ARF, we constructed a point mutation in ARF1, Q71I, which was predicted to slow the rate of GTP hydrolysis. We demonstrate that Q71I, in contrast to wild type ARF1, exhibits a 2-3-fold increase in the half-life of ARF-GTP and is able to promote stable coatomer binding to Golgi membranes in the presence of GTP in vitro.

Purification and Characterization of an l-Aminopeptidase from Pseudomonas putida ATCC 12633.

An l-aminopeptidase of Pseudomonas putida, used in an industrial process for the hydrolysis of d,l-amino acid amide racemates, was purified to homogeneity. The highly l-enantioselective enzyme resembled thiol reagent-sensitive alkaline serine proteinases and was strongly activated by divalent cations. It possessed a high substrate specificity for dipeptides and alpha-H amino acid amides, e.

Localization of TGN38 to the trans-Golgi network: involvement of a cytoplasmic tyrosine-containing sequence.

Protein localization to the TGN was investigated by examining the subcellular distribution of chimeric proteins in which the cytoplasmic and/or transmembrane domains of the TGN protein, TGN38, were substituted for the analogous domains of the plasma membrane protein, Tac. Using immunofluorescence and immunoelectron microscopy, the COOH-terminal cytoplasmic domain of TGN38 was found to be sufficient for localization of the chimeric proteins to the TGN. Deletion analysis identified an 11-amino acid segment containing the critical sequence, YQRL, as being sufficient for TGN localization.

Peptide-induced stabilization and intracellular localization of empty HLA class I complexes.

The human cell line T2 has been reported to be class I assembly deficient, and accordingly expresses reduced amounts of HLA-A2 and no HLA-B5 at the cell surface. By immunoblotting we observe the steady-state class I heavy chain levels of T2 to be near normal when compared with the identical class I alleles of the wild-type cell line T1. In pulse chase experiments, formation of heavy chain beta 2-microglobulin complexes is observed for both HLA-A2 and HLA-B5.

Polarized expression of major histocompatibility complex class I molecules in human endometrial and endocervical epithelial cells.

The localization of major histocompatibility complex (MHC) class I molecules in human endometrial and endocervical epithelial cells was studied with an immunogold technique on ultrathin cryosections. At the cell surface, MHC class I molecules are delivered solely to the basolateral plasma membrane in human epithelia. Therefore, only the basolateral domain of epithelial cells is capable of presenting antigen to MHC class I-restricted cytotoxic T cells.

Cytotoxic T lymphocyte granules are secretory lysosomes, containing both perforin and granzymes.

Cytotoxic T lymphocytes (CTL) contain granules that are exocytosed during specific interaction with target cells (TC). In this process, the granule contents, including the lethal protein perforin, as well as granzymes, a family of serine esterases, are delivered to the TC. Information regarding the routing of these proteins towards the granule and their exact localization within the granule is of primary importance to resolve the mechanism of granule-mediated TC killing.

Segregation of MHC class II molecules from MHC class I molecules in the Golgi complex for transport to lysosomal compartments.

Traffic of MHC molecules dictates the source of peptides that are presented to T cells. The intracellular distribution of MHC class I and class II molecules reflects the dichotomy in presentation of antigen from endogenous and exogenous origin, respectively. In human B lymphoblastoid cells, class I molecules are present in compartments constituting the biosynthetic pathway, whereas class II molecules enter structures related to lysosomes during their biosynthesis.

CD63 antigen. A novel lysosomal membrane glycoprotein, cloned by a screening procedure for intracellular antigens in eukaryotic cells.

To clone the CD63 antigen, originally described as a blood platelet activation marker, we adapted the expression cloning procedure of Seed and Aruffo (Seed, B., and Aruffo, A. (1987) Proc.

Interaction of chondroitin sulfate with perforin and granzymes of cytolytic T-cells is dependent on pH.

Cytolytic T-lymphocytes (CTL) harbor cytoplasmic granules containing the lytic, pore-forming protein perforin, a family of serine proteases designated granzymes, and proteoglycans as major constituents. Growth of CTL lines in the presence of PNP-xyloside completely inhibited the glycosylation of the granule-associated chondroitin sulfate A type proteoglycans. Only short glycosaminoglycan molecules were detected.

The biosynthetic pathway of MHC class II but not class I molecules intersects the endocytic route.

We studied the intracellular traffic and subcellular distribution of MHC class I and class II antigens in comparison with a recycling surface glycoprotein, the transferrin receptor (Tfr), in the human lymphoblastoid cell line JY. No internalization was detectable for class I molecules. Class II molecules were internalized but did not recycle.

Human epidermal Langerhans cells undergo profound morphologic and phenotypical changes during in vitro culture.

Morphology, phenotype, and enzyme activity of highly enriched (80%) unlabeled human epidermal Langerhans cells (LC) have been studied, with emphasis on changes during a short-term culture of three days in vitro. All freshly isolated LC contained Birbeck granules and expressed high levels of CD1a, CD1c, and MHC class II molecules HLA-DR, -DP, and -DQ. They have a weak to moderate expression of RFD1, C3biR, Fc gamma R, p 150/95, MHC class I molecules HLA-ABC, and of the adhesion molecules LFA-3 and ICAM-1, whereas no expression of LFA-1 and several monocyte/macrophage markers were detected.

A new model for lethal hit delivery by cytotoxic T lymphocytes.

Human cytotoxic T lymphocyte (CTL) granules contain an electron-dense core and small membrane vesicles. By immuno-electron microscopy, molecules relevant to CTL-target cell (TC) interactions have been identified on the membranes of the dense core and small vesicles within the granule. Moreover, perforin, the component implicated in the lethal hit, and serine esterases are localized within these granule substructures.

HLA-A- and HLA-B-specific monoclonal antibodies reactive with free heavy chains in western blots, in formalin-fixed, paraffin-embedded tissue sections and in cryo-immuno-electron microscopy.

The mAb HCA2 reacts preferentially with HLA-A locus heavy chains. Its reactivity contrasts with that of HC10, a mAb with more pronounced specificity for HLA-B and -C heavy chains. Both HCA2 and HC10 were raised against free class I heavy chains of HLA-A and -B antigens respectively, to obtain mAbs that would still react with denatured class I antigens, as they occur in Western blotting, conventional light microscopical analysis of formalin-fixed, paraffin-embedded sections, and cryo-immuno-electron microscopy.