Characteristics of the tyrosine recognition signal for internalization of transmembrane surface glycoproteins.

A tyrosine residue in the cytoplasmic domain of a class of cell surface receptors is necessary, but not sufficient, for internalization through coated pits. To identify the amino acid context enabling a tyrosine to serve as a signal for endocytosis, we mutated the short cytoplasmic domain of a mutant influenza virus hemagglutinin that is competent for internalization, HA-Y543, and determined the effect of each change on internalization. From these results and a comparison of sequences of other proteins recognized by coated pits, a "tyrosine internalization signal" was proposed. Site-directed mutagenesis was employed to insert complete, or incomplete "tyrosine internalization signals" into the cytoplasmic domain of a protein normally not endocytosed, human glycophorin A. Only the complete signal caused internalization of mutant glycophorins by coated pits. The signal is formed by a short amino acid sequence, with polar or basic residues preferred at certain positions on either side of the tyrosine. Amino acids, which in proteins of known structure are frequently found in turns, are clustered near the tyrosine on the side of the signal nearest the transmembrane domain.

PtK1 cells contain a nondiffusible, dominant factor that makes the Golgi apparatus resistant to brefeldin A.

Brefeldin A (BFA) was shown in earlier studies of numerous cell types to inhibit secretion, induce enzymes of the Golgi stacks to redistribute into the ER, and to cause the Golgi cisternae to disappear. Here, we demonstrate that the PtK1 line of rat kangaroo kidney cells is resistant to BFA. The drug did not disrupt the morphology of the Golgi complex in PtK1 cells, as judged by immunofluorescence using antibodies to 58- (58K) and 110-kD (beta-COP) Golgi proteins, and by fluorescence microscopy of live cells labeled with C6-NBD-ceramide. In addition, BFA did not inhibit protein secretion, not alter the kinetics or extent of glycosylation of the vesicular stomatitis virus (VSV) glycoprotein (G-protein) in VSV-infected PtK1 cells. To explore the mechanism of resistance to BFA, PtK1 cells were fused with BFA-sensitive CV-1 cells that had been infected with a recombinant SV-40 strain containing the gene for VSV G-protein and, at various times following fusion, the cultures were exposed to BFA. Shortly after cell fusion, heterokaryons contained one Golgi complex associated with each nucleus. Golgi membranes derived from CV-1 cells were sensitive to BFA, whereas those of PtK1 origin were BFA resistant. A few hours after fusion, most heterokaryons contained a single, large Golgi apparatus that was resistant to BFA and contained CV-1 galactosyltransferase. In unfused cells that had been perforated using nitrocellulose filters, retention of beta-COP on the Golgi was optimal in the presence of cytosol, ATP, and GTP. In perforated cell models of the BFA-sensitive MA104 line, BFA caused beta-COP to be released from the Golgi complex in the presence of nucleotides, and either MA104 or PtK1 cytosol. In contrast, when perforated PtK1 cells were incubated with BFA, nucleotides, and cytosol from either cell type, beta-COP remained bound to the Golgi complex. We conclude that PtK1 cells contain a nondiffusible factor, which is located on or very close to the Golgi complex, and confers a dominant resistance to BFA. It is possible that this factor is homologous to the target of BFA in cells that are sensitive to the drug.

Evidence that phospholipase D mediates ADP ribosylation factor-dependent formation of Golgi coated vesicles.

Formation of coatomer-coated vesicles from Golgi-enriched membranes requires the activation of a small GTP-binding protein, ADP ribosylation factor (ARF). ARF is also an efficacious activator of phospholipase D (PLD), an activity that is relatively abundant on Golgi-enriched membranes. It has been proposed that ARF, which is recruited onto membranes from cytosolic pools, acts directly to promote coatomer binding and is in a 3:1 stoichiometry with coatomer on coated vesicles. We present evidence that cytosolic ARF is not necessary for initiating coat assembly on Golgi membranes from cell lines with high constitutive PLD activity. Conditions are also described under which ARF is at most a minor component relative to coatomer in coated vesicles from all cell lines tested, including Chinese hamster ovary cells. Formation of coated vesicles was sensitive to ethanol at concentrations that inhibit the production of phosphatidic acid (PA) by PLD. When PA was produced in Golgi membranes by an exogenous bacterial PLD, rather than with ARF and endogenous PLD, coatomer bound to Golgi membranes. Purified coatomer also bound selectively to artificial lipid vesicles that contained PA and phosphatidylinositol (4,5)-bisphosphate (PIP2). We propose that activation of PLD and the subsequent production of PA are key early events for the formation of coatomer-coated vesicles.

Phosphatidic acid formation by phospholipase D is required for transport from the endoplasmic reticulum to the Golgi complex.

BACKGROUND: Lipid molecules may play a regulatory role in the secretory pathway of mammals and yeast. The lipid hydrolase phospholipase D (PLD) is one candidate for mediating regulation of secretion, based on the location of this enzyme and its requirements for activation. RESULTS: We found that primary alcohols, which block formation of phosphatidic acid (PA) by PLD, inhibited the transport of two different viral glycoproteins from the endoplasmic reticulum to the Golgi complex in Chinese hamster ovary cells. Corresponding secondary alcohols, which are much less potent in blocking PA formation, were also less effective in blocking transport of the glycoproteins. The block in glycoprotein transport imposed by primary alcohols was reversed when PA, in the form of liposomes, was exogenously supplied to the culture medium. CONCLUSIONS: We suggest that the earliest site of regulation of membrane transport by PLD is within the intermediate compartment between the endoplasmic reticulum and the Golgi complex.

A fluorescent lipid analogue can be used to monitor secretory activity and for isolation of mammalian secretion mutants.

The use of reporter proteins to study the regulation of secretion has often been complicated by posttranslational processing events that influence the secretion of certain proteins, but are not part of the cellular mechanisms that specifically regulate secretion. This has been a particular limitation for the isolation of mammalian secretion mutants, which has typically been a slow process. To provide a reporter of secretory activity independent of protein processing events, cells were labeled with the fluorescent lipid analogue C5-DMB-ceramide (ceramide coupled to the fluorophore boron dipyrromethene difluoride) and its secretion was followed by fluorescence microscopy and fluorescence-activated cell sorting. Brefeldin A, which severely inhibits secretion in Chinese hamster ovary cells, blocked secretion of C5-DMB-ceramide. At high temperature, export of C5-DMB-ceramide was inhibited in HRP-1 cells, which have a conditional defect in secretion. Using C5-DMB-ceramide as a reporter of secretory activity, several different pulse-chase protocols were designed that selected mutant Chinese hamster ovary cells that were resistant to the drug brefeldin A and others that were defective in the transport of glycoproteins to the cell surface. Mutant cells of either type were identified in a mutagenized population at a frequency of 10(-6). Thus, the fluorescent lipid C5-DMB-ceramide can be used as a specific marker of secretory activity, providing an efficient, general approach for isolating mammalian cells with defects in the secretory pathway.

Correlative Live Cell and Super Resolution Imaging of Autophagosome Formation.

Autophagy is a highly dynamic intracellular process involving interactions between protein complexes and membranes. Direct observation of these components in living cells provides information on how they interact and when and where they are involved in the autophagy pathway. This chapter provides an overview of methods used to acquire images of fluorescently labeled components of the autophagy pathway in living cells using wide-field microscopy. Due to the diffraction-limited nature of this technique further details are provided on how to acquire postfixation correlative super resolution images from the same cells that have previously been imaged live. Combining these techniques offers an opportunity to follow the processes of autophagy in living cells with unprecedented detail.

Length, mass, and denaturation of double-stranded RNA molecules compared with DNA.

The contour lengths of linear, double-stranded (ds) RNAs from mycovirus PcV and Pseudomonas bacteriophage phi 6 have been measured with samples prepared for the electron microscope from 0.05 to 0.5 M NH4Cl solutions. A linear dependence of contour length on the logarithm of ionic strength was found and compared with that of dsDNA (pBR322, linearized and open-circular forms). Conditions for molecular weight determinations of any natural dsRNA by electron microscopy have been established, and the method has been calibrated with phi 6 dsRNA of known nucleotide sequence. The results imply that dsRNA in 0.20 M NH4Cl solution has a rise per basepair of 0.271 nm, which is shorter than that in the A-conformation (4%) and in the A'-conformation (10%). The thermal behavior of dsRNA in terms of melting temperature and exhibition of fine structure of melting curves was found to be generally similar to that of dsDNA, as expected from the literature. Folding of dsRNA in ethanolic solution was similar to that of dsDNA. However, in contrast to dsDNA, coiled coils could not be induced by ethanol, which is consistent with dsRNA being stiffer than dsDNA. Concerning dsDNA, the results show that a contraction in rise per basepair by 0.1 nm is coupled with an increase in the winding angle between basepairs by 0.47 degrees, as qualitatively predicted by polyelectrolyte theory.

Immunolocalisation of phospholipase D1 on tubular vesicular membranes of endocytic and secretory origin.

We have examined the localisation of overexpressed phospholipase D1 (PLD1) using antibodies against its amino- and carboxyl-terminal domains. PLD1 overexpressed in COS-7 cells showed variable distribution by immunofluorescence but was mainly in punctate structures in the perinuclear region and at the plasma membrane. Downregulation by an anti-sense plasmid resulted in almost exclusively perinuclear distribution in punctate structures that contained immunoreactivity for the endogenous KDEL receptor and the early endosomal antigen EEA1 protein. Influenza haemagglutinin (HA) and HA-derived mutants designed to locate primarily to secretory or endocytic membranes were present in PLD1-positive membranes. Immunofluorescence analysis in permanent CHO cell lines that express PLD1 inducibly confirmed the presence of PLD1 on both endocytic and secretory membranes. Analysis of PLD1 distribution by immunocytochemistry and electron microscopy of intact CHO cells and of isolated membranes revealed that PLD1 was present in tubulovesicular elements and multivesicular bodies. Some of these were close to the Golgi region whereas others stained positive for endocytic cargo proteins. Morphometric analysis assigned the majority of PLD1 immunoreactivity on endosomal membranes and a smaller amount on membranes of secretory origin. PLD1, via signals that are currently not understood, is capable of localising in tubulovesicular membranes of both endocytic and secretory origin.

Phospholipase D as an effector for ADP-ribosylation factor in the regulation of vesicular traffic.

A mammalian phospholipase D (PLD) activity that is stimulated by ADP-ribosylation factor (ARF) has been identified in Golgi-enriched membrane fractions. This activity is due to the PLD1 isoform and evidence from several laboratories indicates that PLD1 is important for the polymerization of vesicle coat proteins on membranes. When expressed in Chinese hamster ovary cells, PLD1 localized to dispersed small vesicles that overlapped with the location of the ERGIC53 protein, a marker for the endoplasmic reticulum (ER)-Golgi intermediate compartment. Cells having increased PLD1 expression had accelerated anterograde and retrograde transport between the ER and Golgi. Membranes from cells having elevated PLD1 activity bound more COPI, ARF, and ARF-GTPase activating protein. These membranes also produced more COPI vesicles than did membranes from control cells. It is likely that PLD1 participates in both positive and negative feedback regulation of the formation of COPI vesicles and is important for controlling the rate of this process.

Lipid phosphate phosphatases and lipid phosphate signalling.

Mammalian LPPs (lipid phosphate phosphatases) are integral membrane proteins that belong to a superfamily of lipid phosphatases/phosphotransferases. They have broad substrate specificity in vitro, dephosphorylating PA (phosphatidic acid), S1P (sphingosine 1-phosphate), LPA (lysophosphatidic acid) etc. Their physiological role may include the attenuation of S1P- and LPA-stimulated signalling by virtue of an ecto-activity (i.e. dephosphorylation of extracellular S1P and LPA), thereby limiting the activation of LPA- and S1P-specific G-protein-coupled receptors at the cell surface. However, our recent work suggests that an intracellular action of LPP2 and LPP3 may account for the reduced agonist-stimulated p42/p44 mitogen-activated protein kinase activation of HEK-293 (human embryonic kidney 293) cells. This may involve a reduction in the basal levels of PA and S1P respectively and the presence of an early apoptotic phenotype under conditions of stress (serum deprivation). Additionally, we describe a model whereby LPP2, but not LPP3, may be functionally linked to the phospholipase D1-derived PA-dependent recruitment of sphingosine kinase 1 to the perinuclear compartment. We also consider the potential regulatory mechanisms for LPPs, which may involve oligomerization. Lastly, we highlight many aspects of the LPP biology that remain to be fully defined.

Signalling molecules and the regulation of intracellular transport.

A variety of signalling molecules has been implicated over the past 8 years in the regulation of intracellular transport pathways. Those molecules include heterotrimeric GTP binding proteins, members of the protein kinase C family, and members of the Rho subfamily of small GTPases. Until recently, no common theme among the three classes of regulators was apparent. The finding that all three can influence the activity of phospholipase D (PLD), and the fact that members of the Arf subfamily of GTPases (with established roles in intracellular transport) are potent activators of PLD suggests the hypothesis that PLD is a focal point for integration of cellular responses to hormone signalling and for membrane homeostasis. Work during the past 2 years is beginning to uncover some transport pathways where PLD involvement is inferred. It is proposed that, if signalling is required to monitor and adjust transport rates to and from the various membrane organelles, the most economical way to achieve this would be to regulate recycling and allow the concentration of cargo receptors to determine forward transport.

The dodecahedral framework of the bacteriophage phi 6 nucleocapsid is composed of protein P1.

The outer layer of the bacteriophage phi 6 nucleocapsid (NC) was removed by EDTA and reassociated with the core in the presence of Ca2+ or Mg2+. The core was relatively inaccessible to trypsin digestion, was composed of protein P1, and was in the dodecahedral framework reported previously. (H.T. Steely, Jr., and D. Lang, J. Virol. 51:479-483, 1984; Y. Yang and D. Lang, J. Virol. 51:484-488, 1984). The double-stranded RNA genome became RNase sensitive after EDTA treatment of the nucleocapsid.

Modification of catalytically active phospholipase D1 with fatty acid in vivo.

Phospholipase D1 (PLD1) was covalently labeled with 3H when expressed transiently in COS cells and immunoprecipitated following labeling of the cells with [3H]palmitate. Labeling of PLD1 was abolished by treatment with hydroxylamine at neutral pH, indicating that the fatty acid is linked via thioester to the enzyme. In pulse-chase studies the label persisted over a 3-h chase, indicating a slow rate of turnover. A catalytically inactive point mutant of PLD1 that changes serine at position 911 to alanine (S911A) was partially but not entirely redistributed to the cytosol, and it contained no detectable palmitate label. Similarly, N- and C-terminal domain fragments of the protein, encompassing in combination the entire coding region and all expressed to levels comparable with the wild type protein, showed no label with palmitate. Treatment of immunoprecipitated PLD1 with hydroxylamine diminished catalytic activity to background levels in a dose response manner that paralleled the removal of label from [3H]palmitate-labeled protein. We suggest that modification of PLD1 with palmitate is related to its catalytic activity and may be an important requirement for the function of this enzyme.

Recruitment of hepatocyte nuclear factor 4 into specific intranuclear compartments depends on tyrosine phosphorylation that affects its DNA-binding and transactivation potential.

Hepatocyte nuclear factor 4 (HNF-4) is a prominent member of the family of liver-enriched transcription factors, playing a role in the expression of a large number of liver-specific genes. We report here that HNF-4 is a phosphoprotein and that phosphorylation at tyrosine residue(s) is important for its DNA-binding activity and, consequently, for its transactivation potential both in cell-free systems and in cultured cells. Tyrosine phosphorylation did not affect the transport of HNF-4 from the cytoplasm to the nucleus but had a dramatic effect on its subnuclear localization. HNF-4 was concentrated in distinct nuclear compartments, as evidenced by in situ immunofluorescence and electron microscopy. This compartmentalization disappeared when tyrosine phosphorylation was inhibited by genistein. The correlation between the intranuclear distribution of HNF-4 and its ability to activate endogenous target genes demonstrates a phosphorylation signal-dependent pathway in the regulation of transcription factor activity.

In vitro assembly of the outer shell of bacteriophage phi 6 nucleocapsid.

Following dissociation of bacteriophage phi 6 nucleocapsid (NC) by EDTA, a particle composed of protein P8 and corresponding to the outer shell of the NC was assembled in vitro in the presence of Ca2+ and Mg2+. Assembly was obtained from soluble protein constituents above 100 micrograms/ml and was optimal within a temperature range of 22-30 degrees. Assembly did not require the presence of genomic RNA. Crosslinking results of intact NCs and in vitro-assembled outer shells suggested that protein P8 dimers are the structural subunits of the shell. Analysis of the assembly kinetics by electron microscopy suggested that ring-like particles of uniform size, packed in flat hexagonal arrays, are intermediates in outer shell assembly.

Action of brefeldin A blocked by activation of a pertussis-toxin-sensitive G protein.

In many mammalian cells brefeldin A interferes with mechanisms that keep the Golgi appartus separate from the endoplasmic reticulum. The earliest effect of brefeldin A is release of the coat protein beta-COP from the Golgi. This release is blocked by pretreatment with GTP-gamma S or AlF4- (ref. 12). The AlF4- ion activates heterotrimeric G proteins but not proteins of the ras superfamily, suggesting that a heterotrimeric G protein might control membrane transfer from the endoplasmic reticulum to the Golgi. We report here that mastoparan, a peptide that activates heterotrimeric G proteins, promotes binding of beta-COP to Golgi membranes in vitro and antagonizes the effect of brefeldin A on beta-COP in perforated cells and on isolated Golgi membranes. This inhibition is greatly diminished if cells are pretreated with pertussis toxin before perforation. Thus, a heterotrimeric G protein of the Gi/Go subfamily regulates association of coat components with Golgi membranes.

Phospholipase D is present on Golgi-enriched membranes and its activation by ADP ribosylation factor is sensitive to brefeldin A.

ADP ribosylation factor (ARF) is a small guanosine triphosphate (GTP)-binding protein that regulates the binding of coat proteins to membranes and is required for several stages of vesicular transport. ARF also stimulates phospholipase D (PLD) activity, which can alter the lipid content of membranes by conversion of phospholipids into phosphatidic acid. Abundant PLD activity was found in Golgi-enriched membranes from several cell lines. Golgi PLD activity was greatly stimulated by ARF and GTP analogs and this stimulation could be inhibited by brefeldin A (BFA), a drug that blocks binding of ARF to Golgi membranes. Furthermore, in Golgi membranes from BFA-resistant PtK1 cells, basal PLD activity was high and not stimulated by exogenous ARF or GTP analogs. Thus, ARF activates PLD on the Golgi complex, suggesting a possible link between transport events and the underlying architecture of the lipid bilayer.

Fatty acylation of phospholipase D1 on cysteine residues 240 and 241 determines localization on intracellular membranes.

We have reported previously that phospholipase D1 (PLD1) is labeled specifically with [(3)H]palmitate following transient expression and immunoprecipitation and that this modification appeared important both for membrane localization and catalytic activity. In this work we identify by mutagenesis that the acylation sites on PLD1 are cysteine residues 240 and 241, with the cysteine at position 241 accounting for most but not all of the modification. Replacement of both cysteine residues with either serines or alanines resulted in a mutant protein that contained undetectable [(3)H]palmitate. In comparison with the wild type protein, the double mutant showed reduced catalytic activity in vivo, whereas its activity in vitro was unchanged. In addition, the localization of the double mutant was altered in comparison with the wild type protein, whereas wild type PLD1 is primarily on intracellular membranes and on punctate structures, the double mutant was on plasma membrane. Because cysteines 240 and 241 lie within a putative pleckstrin homology domain of PLD1, it is likely that fatty acylation on these residues modulates the function of the PLD1 pleckstrin homology domain.

Phospholipase D1 and potential targets of its hydrolysis product, phosphatidic acid.

Phospholipase D (PLD) hydrolyses phosphatidylcholine into phosphatidic acid (PA) and choline. Our work aims to understand the properties of PLD1, and to identify downstream targets of PA. In one set of projects, we have focused on membrane-targeting mechanisms and have proposed a hierarchy of signals that allows PLD1 to localize to intracellular membranes. These signals involve a functional pleckstrin homology (PH) domain and its fatty acylation on two adjacent cysteine residues. A nearby Phox homology (PX) domain may modulate the function of the fatty acylated PH domain. This complex array of signals is probably necessitated by the targeting of PLD1 to multiple endocytic and secretory membranes under basal and signal-dependent conditions. In another set of projects, we have used chemically synthesized PA coupled to a solid support in order to identify proteins that interact with this phospholipid. Several proteins have emerged from this screen as potential targets. Some (e.g. ADP-ribosylation factor, coatomer beta subunit) are involved in trafficking and their PA affinity can be understood in terms of their regulated cycling on and off membranes during rounds of transport. Others (sphingosine 1-phosphate kinase and PtdIns4 P 5-kinase) are implicated in pathways that also involve PLD activation. Others still are novel proteins (brain-specific neurochondrin) whose affinity for PA may contribute to an understanding of their cellular function.