Interaction of the bovine papillomavirus E6 protein with the clathrin adaptor complex AP-1.

The E6 gene of the bovine papillomavirus type 1 (BPV-1) is expressed in fibropapillomas caused by BPV-1 and in tissue culture cells transformed by BPV-1. It encodes one of the two major oncoproteins of BPV-1. In this study, we demonstrate an interaction between the BPV-1 E6 protein and AP-1, the TGN (trans-Golgi network)-specific clathrin adaptor complex. AP-1 is a four-subunit protein complex required for clathrin-mediated cellular transport from the TGN. The AP-1/E6 interaction was observed in vitro and in cells. The E6 binding site on AP-1 was mapped to the N-terminal trunk domain of the gamma subunit. BPV-1 E6 preferentially associated with membrane-bound AP-1 in cells but not with free cytosolic AP-1. BPV-1 E6 was further shown to be recruited to isolated Golgi membranes and to copurify with clathrin-coated vesicles. The recruitment of BPV-1 E6 to Golgi membranes was AP-1 independent, but the E6 interaction with AP-1 was required for its association with clathrin-coated vesicles. Furthermore, AP-1 proteins could compete with BPV-1 E6 for binding to Golgi membranes, suggesting that the recruitment of BPV-1 E6 and AP-1 to Golgi membranes involves a common factor. Taken together, our results suggest that cytosolic BPV-1 E6 is first recruited to the TGN, where it is then recognized by membrane-bound AP-1 and subsequently recruited into TGN-derived clathrin-coated vesicles. We propose that BPV-1 E6, through its interaction with AP-1, can affect cellular processes involving clathrin-mediated trafficking pathway.

Parallel dimers and anti-parallel tetramers formed by epidermal growth factor receptor pathway substrate clone 15.

The recently discovered localization of epidermal growth factor receptor pathway substrate clone 15 (Eps15) to plasma membrane clathrin-coated pits and its constitutive association with the endocytic clathrin adaptor protein complex, AP-2, strongly suggest that Eps15 has an important role in the pathway of clathrin-dependent endocytic traffic. We report here that Eps15 forms dimers and tetramers of distinct shape. The Eps15 dimer is an elongated molecule, 32 nm in length. There is a globular "head" at one end of the molecule and an extended "stalk" of 25 nm which is kinked at about 17 nm away from the head. In the Eps15 dimer, two subunits are arranged parallel to each other, so that the head corresponds to two side by side copies of the N-terminal region I, which contains the three Eps15 homology domains. The proximal part of the stalk is the coiled-coil central region II containing 20 heptad repeats. The kink is at the boundary between region II and the C-terminal region III, which contains the AP-2 binding site, 15 aspartic-proline-phenylalanine repeats, and proline-rich Src homology domain ligand sites. The Eps15 tetramer has a "dumbbell" shape, approximately 31 nm in length; it is formed by the anti-parallel association of two Eps15 dimers. Formation of these Eps15 tetramers appears to require contacts between regions I of one dimer and regions III of a second apposing dimer. The extended shapes of the Eps15 dimers and tetramers suggest how Eps15 oligomers are located in the clathrin coat. We discuss the implications for accessibility to partners and for proposed functions of Eps15.

Wortmannin alters the transferrin receptor endocytic pathway in vivo and in vitro.

Treatment with the phosphatidylinositol 3-kinase inhibitor wortmannin promotes approximately 30% decrease in the steady-state number of cell-surface transferrin receptors. This effect is rapid and dose dependent, with maximal down-regulation elicited with 30 min of treatment and with an IC50 approximately 25 nM wortmannin. Wortmannin-treated cells display an increased endocytic rate constant for transferrin internalization and decreased exocytic rate constants for transferrin recycling. In addition to these effects in vivo, wortmannin is a potent inhibitor (IC50 approximately 15 nM) of a cell-free assay that detects the delivery of endocytosed probes into a common compartment. Inhibition of the in vitro assay involves the inactivation of a membrane-associated factor that can be recruited onto the surface of vesicles from the cytosol. Its effects on the cell-free assay suggest that wortmannin inhibits receptor sorting and/or vesicle budding required for delivery of endocytosed material to "mixing" endosomes. This idea is consistent with morphological changes induced by wortmannin, which include the formation of enlarged transferrin-containing structures and the disruption of the perinuclear endosomal compartment. However, the differential effects of wortmannin, specifically increased transferrin receptor internalization and inhibition of receptor recycling, implicate a role for phosphatidylinositol 3-kinase activity in multiple sorting events in the transferrin receptor's membrane traffic pathway.

Role of the regulatory domain of the EGF-receptor cytoplasmic tail in selective binding of the clathrin-associated complex AP-2.

BACKGROUND: After stimulation of a cell by the mitogenic epidermal growth factor (EGF), the EGF receptor (EGF-R) is cleared from the cell surface in order to turn off receptor signaling. This internalization is mediated via clathrin-coated pits and coated vesicles, and ultimately the receptors are delivered to the lysosome and destroyed. It is believed that clathrin-associated protein complexes or adaptors (APs) link the entrapment of EGF-R and other nutrient and growth-factor receptors to the formation of the clathrin-coated pit. Two classes of APs are known--AP-2, found at the plasma membrane, and AP-1, found in the trans-Golgi network. Activated EGF-R associates with AP-2s at the plasma membrane, but the mechanism responsible for this association is not known. Here, we investigate, in vivo and in vitro, three aspects of the interaction between APs and EGF-R: firstly, we ask whether EGF-R at the plasma membrane distinguishes between AP-1 and AP-2; secondly, we ask which part of the receptor's cytoplasmic tail is responsible for binding; finally, we ask whether autophosphorylation by EGF-R is essential for the interaction. RESULTS: We demonstrate that EGF-R displays a selective association for AP-2 over AP-1 in vivo, and that this preferential interaction can also be detected using surface plasmon resonance in vitro. Using a truncated mutant and a kinase-dead mutant of EGF-R, we show that the regulatory domain of the cytoplasmic tail is essential for the recruitment of AP-2 in vivo and that this domain is required for association between purified AP-2 and EGF-R in vitro. Finally, we demonstrate, in vivo and in vitro, that tyrosine auto-phosphorylation by the receptor is not an essential pre-condition for the recruitment of AP-2. CONCLUSIONS: EGF-R binds selectively to AP-2s, and the regulatory domain of its cytoplasmic tail is required for this interaction. The lack of correlation between receptor autophosphorylation and AP-2 recruitment suggests that activation of the EGF-R kinase stimulates endocytosis by the phosphorylation of a factor distinct from EGF-R itself, as also proposed by others based on experiments measuring receptor traffic and entrapment.

Messenger RNAs expressed in intestine of adult but not baby rabbits. Isolation of cognate cDNAs and characterization of a novel brush border protein with esterase and phospholipase activity.

Using a subtractive hybridization method, we have cloned cDNAs corresponding to 10 different mRNAs which share the property of being expressed in the intestine of adult but not baby rabbits. Four could be identified as coding for previously known gene products (sucrase-isomaltase, a glutathione S-transferase, a cytochrome P450, and a long form of ferritin mRNA), while six code for previously unknown proteins. One clone, AdRab-B, codes for a protein of 1458 amino acids, including (i) a putative signal sequence at the NH2 terminus, (ii) four internal repeats, 308-346 amino acids in length, (iii) a hydrophobic stretch near the COOH terminus, which represents a potential membrane anchor, and (iv) a short hydrophilic stretch at the very COOH terminus. The corresponding protein was studied with the aid of antibodies prepared against polypeptides expressed from segments of the cDNA in Escherichia coli. The protein was shown to be proteolytically processed in the intestine (but not when expressed in COS cells) and to be targeted to the brush border membrane of the enterocytes. Finally, the protein was found to have esterase and phospholipase A/lysophospholipase activity.

Distinct pathways for basolateral targeting of membrane and secretory proteins in polarized epithelial cells.

Polarized epithelial cells target distinct sets of membrane and secretory proteins to their apical and basolateral domains. Here we examine whether constitutively secreted and membrane proteins that are bound for the same domain share the same carrier vesicles. To address the issue, differential effects of microtubule depolymerization on basolateral protein targeting in the polarized Madin-Darby canine kidney II cell line were studied. We find that the basolateral insertion of the active, ouabain-binding Na+,K(+)-ATPase and of a set of very late antigen integrins is little affected by microtubule disruption. Under equivalent conditions, the basolateral secretion of the basement membrane protein laminin is strongly suppressed. More specifically, it is demonstrated that microtubules are involved in targeting laminin, but not integrins, from the compartment related to the accumulation of newly synthesized proteins at 20 degrees C (trans-Golgi network) to the basolateral domain. Our study also reveals that laminin associated with basolateral binding sites interacts with those sites only secondarily to secretion. The data provide evidence for a branch in the basolateral targeting pathway, with secreted and membrane proteins loaded into distinct carrier vesicles.

Complete primary structure of human and rabbit lactase-phlorizin hydrolase: implications for biosynthesis, membrane anchoring and evolution of the enzyme.

We report the primary structures of human and rabbit brush border membrane beta-glycosidase complexes (pre-pro-lactase-phlorizin hydrolase, or pre-pro-LPH, EC 3.2.1.23-62), as deduced from cDNA sequences. The human and rabbit primary translation products contain 1927 and 1926 amino acids respectively. Based on the data, as well as on peptide sequences and further biochemical data, we conclude that the proteins comprise five domains: (i) a cleaved signal sequence of 19 amino acids; (ii) a large 'pro' portion of 847 amino acids (rabbit), none of which appears in mature, membrane-bound LPH; (iii) the mature LPH, which contains both the lactase and phlorizin hydrolase activities in a single polypeptide chain; (iv) a membrane-spanning hydrophobic segment near the carboxy terminus, which serves as membrane anchor; and (v) a short hydrophilic segment at the carboxy terminus, which must be cytosolic (i.e. the protein has an Nout-Cin orientation). The genes have a 4-fold internal homology, suggesting that they evolved by two cycles of partial gene duplication. This repetition also implies that parts of the 'pro' portion are very similar to parts of mature LPH, and hence that the 'pro' portion may be a water-soluble beta-glycosidase with another cellular location than LPH. Our results have implications for the decline of LPH after weaning and for human adult-type alactasia, and for the evolutionary history of LPH.

A new approach to high sensitivity differential hybridization.

We describe a new approach to differential hybridization, designed to identify cDNA clones representing rare mRNA species. Duplicate filters carrying a library of cDNA from phorbolmyristate acetate (PMA)-induced EL-4 cells in lambda gt11 were hybridized with high concentrations of unlabeled, cloned, single-stranded cDNA from induced and control EL-4 cells, respectively. Plaques binding single-stranded cDNA were revealed by a second round of hybridization with 35S-labeled DNA complementary to the vector moiety of the single-stranded cDNA. Plaques corresponding to PMA-induced mRNAs occurring at a level of about 1 part in 15,000 were isolated. We believe the method is at least ten times more sensitive than conventional differential hybridization.

Characterisation of the enzyme intermediates of the sarcoplasmic transport ATPase by the use of inhibitors.

1. Based on a detailed reaction scheme of the phosphorylation process of the sarcoplasmic transport ATPase the inhibition mechanisms of benzoctamine, DIO 9, AMP-PNP and of Ca2+-ions at relatively high concentrations (1 approximately 100 microM) were determined. 2. The inhibition mechanisms were analyzed by measuring the gamma-phosphate exchange between ATP and ADP and evaluated by applying conventional and an extended Dixon plot procedures. 3. The kinetic patterns of the inhibition were shown to be compatible with the assumed reaction scheme. 4. Each inhibitor combines with definite intermediates: Benzoctamine with the intermediate species Ca2MgE and Ca2Mg2E-ATP; AMP-PNP with Ca2Mg2E approximately P; DIO 9 with E and MgE and Ca2+ at relatively high concentrations with E. 5. The central intermediate blocked by benzoctamine can partially exist as Ca2Mg2E ADPP-benzoctamine which is detected as phosphoprotein after acid denaturation.

Structure and expression of cloned murine IFN-alpha genes.

The mouse has an interferon-alpha (MuIFN-alpha) gene family containing at least four, and likely more than ten members. A segment of mouse chromosomal DNA and cDNAs encoding murine alpha IFNs have been cloned, and the sequence of two MuIFN-alpha DNAs determined. No intron was found in the chromosomal gene. The two coding sequences produced biologically active IFN when expressed in monkey cells under the control of an SV40 promoter, and in E.coli under the control of the ampicillinase promoter. MuIFN-alpha 1 had no detectable activity on human cells, while MuIFN-alpha 2 was 20% as active on human as on mouse cells.

Structure and expression of human IFN-alpha genes.

Copy DNA (cDNA) was prepared from induced leucocyte poly(A) RNA and cloned in Escherichia coli. IFN-alpha cDNA clones were isolated by subculture cloning with the use of a translation hybridization assay. Definitive identification of the clones was based on the production of an interferon-like protein by the transformed bacteria. Different IFN-alpha cDNAs, with characteristic target cell specificities, were identified. The cloned cDNAs typically encode a mature polypeptide of 166 (or, for IFN-alpha 2, 165) amino acids and a signal sequence of 23 amino acids. A human chromosomal library was screened with IFN cDNA and 17 distinct IFN-alpha-related sequences were isolated and identified, of which 7 proved to be nonallelic authentic genes and 4 pseudogenes; 6 sequences remain to be elucidated. Taking into account the work of Goeddel and his colleagues, 13 non-allelic authentic genes and 6 pseudogenes can be distinguished. In addition, 9 genes believed to be allelic to the 13 authentic genes have been sequenced. The IFN-alpha genes may be classified into two major subfamilies, which diverged at least 33 Ma ago, but perhaps much earlier, if sequence rectification occurred. At least one IFN-alpha gene appears to have resulted by a recombinational event between members of the subfamily I and II. IFN-beta is distantly related to IFN-alpha's and may have diverged from a common ancestor at least 500 Ma ago. Both IFN-alpha and IFN-beta genes differ from most other genes of higher organisms by being devoid of introns. The mouse was found to possess an IFN-alpha gene family of a size similar to that of man; the murine genes also do not have introns. IFN-alpha genes devoid of their signal sequence were joined to prokaryotic promoters to produce the mature interferons in E. coli in high yield. IFN-alpha 2, purified to homogeneity, has been crystallized by T. Unge and B. Strandberg (Uppsala). Hybrid genes consisting of IFN-alpha 1 and IFN-alpha 2 segments were constructed and expressed in E. coli; the target cell specificities of such hybrids were dependent on the arrangement of the segments and were different from those of either parent. The chromosomal gene for HuIFN-alpha 1 was introduced into mouse L cells to study the mechanism of its expression. Correct transcription was only detected after induction (with Newcastle disease virus); expression was transient, with the same kinetics as those of the endogenous mouse IFN mRNA. Natural murine IFNs and human IFN-beta and IFN-gamma are glycosylated. Because E. coli cells transformed with the genes of eukaryotic glycoproteins are not expected to yield correctly glycosylated polypeptides, we prepared lines of hamster cells permanently transformed with hybrid plasmids, which contained an IFN gene linked to the SV40 early promoter, as well as dihydrofolate reductase as a selective marker. After intracellular amplification of the introduced genes, cell lines were obtained which constitutively produced IFN at about 40 000 units ml-1 and could be propagated for at least several months.

Synthesis in E. coli of a polypeptide with human leukocyte interferon activity.

Double-stranded cDNA prepared from the 12S fraction of poly(A) RNA from interferon (IF)-producing human leukocytes was cloned in Escherichia coli using the pBR322 vector. One of the resulting clones had a 910-base pair insert which could hybridise to IF mRNA and was responsible for the production of a polypeptide with biological IF activity. Up to 10,000 units IF activity per g of cells was obtained from some clones.

Hybrid plasmids containing an active thymidine kinase gene of Herpes simplex virus 1.

The gene for the thymidine kinase (TK) of Herpes simplex virus type 1 (HSV-1) is located in the KpnI m and BamHI p fragments of the genome (Wigler et al., Cell 11, 223-232 (1977)). These fragments have been inserted into the EcoRI and BamHI sites, respectively, of plasmid pBR322, and propagated in E.coli. The TK gene contained in the recombinant plasmids was shown to be biologically active when introduced into TK- mouse L cells. Detailed restriction site maps of the BamHI p fragment have been constructed and the approximate location of the TK gene has been determined. Mouse cells transformed with cloned HSV-1 tk+ DNA produced HSV-1-specific thymidine kinase; superinfection with HSV-1 tk- virus increased the level of TK activity tenfold, suggesting that the BamHI p sequences present in transformed cells respond to virus-encoded regulatory gene product(s).