Amiloride-induced stimulation of HCO3− reabsorption in turtle bladder

Amiloride in the mucosal fiuid (at concentrations of 5 · 10^?6 M to 10^?4 M) reversibly stimulates the HCO₃^?-dependent moiety of the short-circuiting current (I_sc) in ouabain-treated turtle bladders bathed by Na-free Ringer solutions with or without Cl_?.This effect is uniquely different from the known inhibitory effect of this agent on Na⁺ transport. Thus, any comprehensive hypothesis on the action of amiloride over a wide dosage-response fange should take into account its effect on HCO₃^? transport.     

Clathrin-coated pits contain an integral membrane protein that binds the AP-2 subunit with high affinity

Coated pits will assemble onto purified plasma membranes that are attached to a poly-L-lysine coated substratum (Moore, M. S., Mahaffey, D. T., Brodsky, F. M., and Anderson, R. G. W. (1987) Science 236, 558-563; Mahaffey, D. T., Moore, M. S., Brodsky, F. M., and Anderson, R. G. W. (1989) J. Cell Biol. 108, 1615-1624). To better understand the assembly reaction, we have purified both clathrin triskelion and AP-2 subunits from bovine brain and assayed for their ability to bind to the cytoplasmic surface of attached membranes. Two types of membranes were analyzed: those washed with a high pH buffer that selectively removes triskelions and those washed with a high salt buffer that removes both the AP-2 and the triskelion subunits. We found that purified AP-2 subunits bind with high affinity (Kd approximately 3 x 10(-8) M) to salt stripped membranes. Binding is saturable and abolished by treating membranes with less than 20 micrograms/ml of elastase. When membranes were treated with elastase before the salt wash and then salt washed and assayed for AP-2 binding, normal binding was seen, which indicates that the presence of clathrin-coated pits protects the binding site from the protease. Membranes that had rebound AP-2 did not bind purified triskelions, even though high pH buffer-washed membranes that bear endogenous AP-2 bound triskelions with high affinity (Kd approximately 3 x 10(-9) M) and supported lattice assembly. We conclude that coated pit assembly is initiated by the binding of AP-2 to an integral membrane protein but that the AP-2 complex must be activated by an unknown process before the coated pit lattice will assemble.     

Interaction of the unique N-terminal region of tyrosine kinase p56lck with cytoplasmic domains of CD4 and CD8 is mediated by cysteine motifs

p56lck, a lymphocyte-specific member of the src family of cytoplasmic protein-tyrosine kinases, is associated noncovalently with the cell surface glycoproteins CD4 and CD8, which are expressed on functionally distinct subpopulations of T cells. Using transient coexpression of p56lck with CD4 or CD8 alpha in COS-7 cells, we show that the unique N-terminal region of p56lck binds to the membrane-proximal 10 and 28 cytoplasmic residues of CD8 alpha and CD4, respectively. Two cysteine residues in each of the critical sequences in CD4, CD8 alpha, and p56lck are required for association. Our results suggest a novel role for cysteine-mediated interactions between unrelated proteins and provide a model for the association of other src-like cytoplasmic kinases with transmembrane proteins.     

Activated T cells and monocytes have characteristic patterns of class II antigen expression

The expression of human histocompatibility class II Ag was measured on activated T cells and monocytes by quantitative mAb binding in direct two-color immunofluorescence. Monocytes activated by IFN-gamma bound an average of 2 x 10(6) DR-specific mAb, 3 x 10(5) DQ-specific mAb, and 7 x 10(5) DP-specific mAb per cell. For T cells activated by anti-CD3, a subpopulation bound 1 x 10(5) DR-specific mAb, 5 x 10(4) DQ-specific mAb and 5 x 10(4) DP-specific mAb per cell. These measurements were obtained after establishing a base line of class II Ag expression on resting B cells and monocytes. Resting B cells and those monocytes that were positive for class II Ag bound identical amounts of mAb; 3 x 10(4) DR-specific mAb, 3 x 10(3) DQ-specific mAb and 2 x 10(4) DP-specific mAb. However, most resting monocytes (75%) expressed only DR Ag. In the process of studying the expression of class II Ag on T cells, it was necessary to define and analyze the activated T cell state. Cell cultures activated with 0.3 ng/ml anti-CD3 had the highest expression of class II Ag on T cells, whereas those activated with 3.0 ng/ml anti-CD3 had the highest expression of IL-2R on T cells. Addition of IL-2 had no further effect on DR Ag expression on T cells but did up-regulate IL-2R expression. Reducing the initial monocyte concentration before activating T cells increased class II Ag expression on T cells without affecting IL-2R expression. The results obtained on T cell activation suggest that perhaps a lymphokine may be made by CD3-activated T cells which induces class II Ag expression on T cells.     

Antigen processing and presentation: close encounters in the endocytic pathway

Stimulation of helper T cells by class II molecules occurs when the class II molecules bind and display peptides derived from foreign antigens that have been endocytosed. The formation of peptide-class II complexes requires antigen degradation and exposure of the peptide-binding site of class II molecules, both of which depend on proteolysis and low pH in the endocytic pathway. This review discusses the role of specific compartments of the endocytic pathway in the generation of antigenic peptides, and in the binding of antigenic peptides to newly synthesized class II molecules and those that are internalized from the cell surface.     

Clathrin assembly protein AP-2 induces aggregation of membrane vesicles: a possible role for AP-2 in endosome formation

We have examined the in vitro behavior of clathrin-coated vesicles that have been stripped of their surface coats such that the majority of the clathrin is removed but substantial amounts of clathrin assembly proteins (AP) remain membrane-associated. Aggregation of these stripped coated vesicles (s-CV) is observed when they are placed under conditions that approximate the pH and ionic strength of the cell interior (pH 7.2, approximately 100 mM salt). This s-CV aggregation reaction is rapid (t1/2 < or = 0.5 min), independent of temperature within a range of 4-37 degrees C, and unaffected by ATP, guanosine-5'-O-(3-thiophosphate), and in particular EGTA, distinguishing it from Ca(2+)-dependent membrane aggregation reactions. The process is driven by the action of membrane-associated AP molecules since partial proteolysis results in a full loss of activity and since aggregation is abolished by pretreatment of the s-CVs with a monoclonal antibody that reacts with the alpha subunit of AP-2. However, vesicle aggregation is not inhibited by PPPi, indicating that the previously characterized polyphosphate-sensitive AP-2 self-association is not responsible for the reaction. The vesicle aggregation reaction can be reconstituted: liposomes of phospholipid composition approximating that found on the cytoplasmic surfaces of the plasma membrane and of coated vesicles (70% L-alpha-phosphatidylethanolamine (type I-A), 15% L-alpha-phosphatidyl-L-serine, and 15% L-alpha-phosphatidylinositol) aggregated after addition of AP-2, but not of AP-1, AP-3 (AP180), or pure clathrin triskelions. Aggregation of liposomes is abolished by limited proteolysis of AP-2 with trypsin. In addition, a highly purified AP-2 alpha preparation devoid of beta causes liposome aggregation, whereas pure beta subunit does not, consistent with results obtained in the s-CV assay which also indicate the involvement of the alpha subunit. Using a fluorescence energy transfer assay we show that AP-2 does not cause fusion of liposomes under physiological solution conditions. However, since the fusion of membranes necessarily requires the close opposition of the two participating bilayers, the AP-2-dependent vesicle aggregation events that we have identified may represent an initial step in the formation and fusion of endosomes that occur subsequent to endocytosis and clathrin uncoating in vivo.     

Folding and trimerization of clathrin subunits at the triskelion hub.

The triskelion shape of the clathrin molecule enables it to form the polyhedral protein network that covers clathrin-coated pits and vesicles. Domains within the clathrin heavy chain that are responsible for maintaining triskelion shape and function were identified and localized. Sequences that mediate trimerization are distal to the carboxyl terminus and are adjacent to a domain that mediates both light chain binding and clathrin assembly. Structural modeling predicts that within this domain, the region of heavy chain-light chain interaction is a bundle of three or four alpha helices. These studies establish a low resolution model of clathrin subunit folding in the central portion (hub) of the triskelion, thus providing a basis for future mutagenesis experiments.     

Can modulators of apolipoproteinB biogenesis serve as an alternate target for cholesterol-lowering drugs?

Understanding the molecular defects underlying cardiovascular disease is necessary for the development of therapeutics. The most common method to lower circulating lipids, which reduces the incidence of cardiovascular disease, is statins, but other drugs are now entering the clinic, some of which have been approved. Nevertheless, patients cannot tolerate some of these therapeutics, the drugs are costly, and/or the treatments are approved for only rare forms of disease. Efforts to find alternative treatments have focused on other factors, such as apolipoproteinB (apoB), which transports cholesterol in the blood stream. The levels of apoB are regulated by endoplasmic reticulum (ER) associated degradation as well as by a post ER degradation pathway in model systems, and we suggest that these events provide novel therapeutic targets. We discuss first how cardiovascular disease arises and how cholesterol is regulated, and then summarize the mechanisms of action of existing treatments for cardiovascular disease. We then review the apoB biosynthetic pathway, focusing on steps that might be amenable to therapeutic interventions.     

A hyper-recombination mutation in S. cerevisiae identifies a novel eukaryotic topoisomerase

A hyper-recombination mutation was isolated that causes an increase in recombination between short repeated delta sequences surrounding the SUP4-omicron gene in S. cerevisiae. The wild-type copy of this gene was cloned by complementation of one of its pleiotropic phenotypes, slow growth. DNA sequence of the clone revealed a 656 amino acid open reading frame capable of encoding a protein homologous to the bacterial type I topoisomerase. No homology was detected with previously identified eukaryotic topoisomerases. Construction of double mutants with either of the two known yeast topoisomerase genes revealed synergistic effects on growth suggesting overlapping functions. Expression of bacterial topoisomerase I in yeast can fully complement the slow growth defect of a null mutation. We have named this locus TOP3 and suggest that it defines a novel eukaryotic topoisomerase gene.