IQGAP1 stimulates actin assembly through the N-WASP-Arp2/3 pathway

IQGAP1 is a conserved modular protein overexpressed in cancer and involved in organizing actin and microtubules in motile processes such as adhesion, migration, and cytokinesis. A variety of proteins have been shown to interact with IQGAP1, including the small G proteins Rac1 and Cdc42, actin, calmodulin, beta-catenin, the microtubule plus end-binding proteins CLIP170 (cytoplasmic linker protein) and adenomatous polyposis coli. However, the molecular mechanism by which IQGAP1 controls actin dynamics in cell motility is not understood. Quantitative co-localization analysis and down-regulation of IQGAP1 revealed that IQGAP1 controls the co-localization of N-WASP with the Arp2/3 complex in lamellipodia. Co-immunoprecipitation supports an in vivo link between IQGAP1 and N-WASP. Pull-down experiments and kinetic assays of branched actin polymerization with N-WASP and Arp2/3 complex demonstrated that the C-terminal half of IQGAP1 activates N-WASP by interacting with its BR-CRIB domain in a Cdc42-like manner, whereas the N-terminal half of IQGAP1 antagonizes this activation by association with a C-terminal region of IQGAP1. We propose that signal-induced relief of the autoinhibited fold of IQGAP1 allows activation of N-WASP to stimulate Arp2/3-dependent actin assembly.     

Fyn is a downstream target of the pleiotrophin/receptor protein tyrosine phosphatase beta/zeta-signaling pathway: regulation of tyrosine phosphorylation of Fyn by pleiotrophin

Pleiotrophin (PTN the protein, Ptn the gene) signals downstream targets through inactivation of its receptor, the transmembrane receptor protein tyrosine phosphatase (RPTP)beta/zeta, disrupting the balanced activity of RPTPbeta/zeta and the activity of a constitutively active tyrosine kinase. As a consequence of the inactivation of RPTPbeta/zeta, PTN stimulates a sharp increase in the levels of tyrosine phosphorylation of the substrates of RPTPbeta/zeta in PTN-stimulated cells. We now report that the Src family member Fyn interacts with the intracellular domain of RPTPbeta/zeta in a yeast two-hybrid system. We further demonstrate that Fyn is a substrate of RPTPbeta/zeta, and that tyrosine phosphorylation of Fyn is sharply increased in PTN-stimulated cells. In previous studies, we demonstrated that beta-catenin and beta-adducin are targets of the PTN/RPTPbeta/zeta-signaling pathway and defined the mechanisms through which tyrosine phosphorylation of beta-catenin and beta-adducin disrupts cytoskeletal protein complexes. We conclude that Fyn is a downstream target of the PTN/RPTPbeta/zeta-signaling pathway and suggest that PTN coordinately regulates tyrosine phosphorylation of beta-catenin, beta-adducin, and Fyn through the PTN/RPTPbeta/zeta-signaling pathway and that together Fyn, beta-adducin, and beta-catenin may be effectors of the previously described PTN-stimulated disruption of cytoskeletal stability, increased cell plasticity, and loss of cell-cell adhesion that are characteristic of PTN-stimulated cells and a feature of many human malignant cells in which mutations have established constitutive expression of the Ptn gene.     

Iron regulatory protein 2 turnover through a nonproteasomal pathway

Iron regulatory protein 2 (IRP2) controls the synthesis of many proteins involved in iron metabolism, and the level of IRP2 itself is regulated by varying the rate of its degradation. The proteasome is known to mediate degradation, with specificity conferred by an iron-sensing E3 ligase. Most studies on the degradation of IRP2 have employed cells overexpressing IRP2 and also rendered iron deficient to further increase IRP2 levels. We utilized a sensitive, quantitative assay for IRP2, which allowed study of endogenous IRP2 degradation in HEK293A cells under more physiologic conditions. We found that under these conditions, the proteasome plays only a minor role in the degradation of IRP2, with almost all the IRP2 being degraded by a nonproteasomal pathway. This new pathway is calcium-dependent but is not mediated by calpain. Elevating the cellular level of IRP2 by inducing iron deficiency or by transfection causes the proteasomal pathway to account for the major fraction of IRP2 degradation. We conclude that under physiological, iron-sufficient conditions, the steady-state level of IRP2 in HEK293A cells is regulated by the nonproteasomal pathway.     

Mechanism of fibre assembly through the chaperone-usher pathway

The chaperone-usher pathway directs the formation of adhesive surface fibres in numerous pathogenic Gram-negative bacteria. The fibres or pili consist exclusively of protein subunits that, before assembly, form transient complexes with a chaperone in the periplasm. In these chaperone:subunit complexes, the chaperone donates one beta-strand to complete the imperfect immunoglobulin-like fold of the subunit. During pilus assembly, the chaperone is replaced by a polypeptide extension of another subunit in a process termed 'donor strand exchange' (DSE). Here we show that DSE occurs in a concerted reaction in which a chaperone-bound acceptor subunit is attacked by another chaperone-bound donor subunit. We provide evidence that efficient DSE requires interactions between the reacting subunits in addition to those involving the attacking donor strand. Our results indicate that the pilus assembly platforms in the outer membrane, referred to as ushers, catalyse fibre formation by increasing the effective concentrations of donor and acceptor subunits.     

On the hepatic mechanism of HDL assembly by the ABCA1/apoA-I pathway

The mechanism for the assembly of HDL with cellular lipid by ABCA1 and helical apolipoprotein was investigated in hepatocytes. Both HepG2 cells and mouse primary culture hepatocytes produced HDL with apolipoprotein A-I (apoA-I) whether endogenously synthesized or exogenously provided. Probucol, an ABCA1 inactivator, inhibited these reactions, as well as the reversible binding of apoA-I to HepG2. Primary cultured hepatocytes of ABCA1-deficient mice also lacked HDL production regardless of the presence of exogenous apoA-I. HepG2 cells secreted apoA-I into the medium even when ABCA1 was inactivated by probucol, but it was all in a free form as HDL production was inhibited. When a lipid-free apoA-I-specific monoclonal antibody, 725-1E2, was present in the culture medium, production of HDL was suppressed, whether with endogenous or exogenously added apoA-I, and the antibody did not influence HDL already produced by HepG2 cells. We conclude that the main mechanism for HDL assembly by endogenous apoA-I in HepG2 cells is an autocrine-like reaction in which apoA-I is secreted and then interacts with cellular ABCA1 to generate HDL.     

The novel cellular mechanism of human 5-HT6 receptor through an interaction with Fyn

The human 5-HT(6) receptor (5-HT(6)R) is one of the latest cloned receptors among the known 5-HT receptors. Its abundant distribution in the limbic region, which participates in the control of mood and emotion and is involved in nervous system diseases such as depression and Alzheimer disease, has caused it to generate much interest. However, the cellular mechanisms of 5-HT(6)R are poorly understood. In the present study we found, using a yeast two-hybrid assay, that the carboxyl-terminal region of 5-HT(6)R interacts with the Fyn-tyrosine kinase. We also determined using a glutathione S-transferase pulldown assay that this interaction was mediated through the SH3 domain of Fyn and confirmed this by co-immunoprecipitation assays in two different transfected cell lines as well as in adult rat brains. Immunocyto(histo)chemistry also showed prominent co-localization between 5-HT(6)R and Fyn in transfected cells and a similar distribution between 5-HT(6)R and Fyn in the rat brain. Based on this interaction, we further examined the modulation of 5-HT(6)R by Fyn and vice versa. In addition, we demonstrated that the activation of 5-HT(6)R activated the extracellular signal-regulated kinase1/2 via an Fyn-dependent pathway. These findings suggest that Fyn may play an important role in 5-HT(6)R- mediated signaling pathways in the central nervous system.     

Early events in the import/assembly pathway of an integral thylakoid protein

The light-harvesting chlorophyll a/b protein (LHCP) is nuclear-encoded and must traverse the chloroplast envelope before becoming integrally assembled into thylakoid membranes. Previous studies implicated a soluble stromal form of LHCP in the assembly pathway, but relied upon assays in which the thylakoid insertion step was intentionally impaired [Cline, K., Fulsom, D. R. and Viitanen, P. V. (1989) J. Biol. Chem. 264, 14225-14232]. Here we have developed a rapid-stopping procedure, based upon the use of HgCl2, to analyze early events of the uninhibited assembly process. With this approach, we have found that proper assembly of LHCP into thylakoids lags considerably behind trans-envelope translocation. During the first few minutes of import, two distinct populations of mature-size LHCP accumulate within the chloroplast. One is the aforementioned soluble stromal intermediate, while the other is a partially (or improperly) assembled thylakoid species. Consistent with precursor/product relationships, both species reach peak levels at a time when virtually none of the imported molecules are correctly assembled. These results confirm and extend our previous interpretation, that upon import, preLHCP is rapidly processed to its mature form, giving rise to a soluble stromal intermediate. They further suggest that the stromal intermediate initially inserts into the thylakoid bilayer in a partially assembled form, which eventually becomes properly assembled into the light-harvesting complex.     

Striatal‐enriched protein tyrosine phosphatase regulates the PTPα/Fyn signaling pathway

The tyrosine kinase Fyn has two regulatory tyrosine residues that when phosphorylated either activate (Tyr⁴²⁰) or inhibit (Tyr⁵³¹) Fyn activity. Within the central nervous system, two protein tyrosine phosphatases (PTPs) target these regulatory tyrosines in Fyn. PTPα dephosphorylates Tyr⁵³¹ and activates Fyn, while STEP (STriatal‐Enriched protein tyrosine Phosphatase) dephosphorylates Tyr⁴²⁰ and inactivates Fyn. Thus, PTPα and STEP have opposing functions in the regulation of Fyn; however, whether there is cross talk between these two PTPs remains unclear. Here, we used molecular techniques in primary neuronal cultures and in vivo to demonstrate that STEP negatively regulates PTPα by directly dephosphorylating PTPα at its regulatory Tyr⁷⁸⁹. Dephosphorylation of Tyr⁷⁸⁹ prevents the translocation of PTPα to synaptic membranes, blocking its ability to interact with and activate Fyn. Genetic or pharmacologic reduction in STEP₆₁ activity increased the phosphorylation of PTPα at Tyr⁷⁸⁹, as well as increased translocation of PTPα to synaptic membranes. Activation of PTPα and Fyn and trafficking of GluN2B to synaptic membranes are necessary for ethanol (EtOH) intake behaviors in rodents. We tested the functional significance of STEP₆₁ in this signaling pathway by EtOH administration to primary cultures as well as in vivo, and demonstrated that the inactivation of STEP₆₁ by EtOH leads to the activation of PTPα, its translocation to synaptic membranes, and the activation of Fyn. These findings indicate a novel mechanism by which STEP₆₁ regulates PTPα and suggest that STEP and PTPα coordinate the regulation of Fyn.

     

The phosphodiesterase 5 inhibitor sildenafil stimulates angiogenesis through a protein kinase G/MAPK pathway

cGMP-degrading pathways have received little attention in the context of angiogenesis. In the present study we set out to determine whether cGMP-specific phosphodiesterase 5 (PDE5) inhibition affects new blood vessel growth. Incubation of chicken chorioallantoic membranes (CAMs) in vivo with sildenafil increased vascular length in a dose-dependent manner. Moreover, incubation of cultured endothelial cells (ECs) with the PDE5 inhibitor promoted proliferation, migration, and organization into tube-like structures. The effects of sildenafil on the angiogenesis-related properties of EC could be blocked by pre-treatment with the soluble guanylyl cyclase (sGC) inhibitor ODQ or the protein kinase G (PKG) I inhibitor DT-3. In addition, over-expression of sGC in EC led to an enhanced growth and migratory response to sildenafil. To study the signaling pathways implicated in the sildenafil-stimulated angiogenic responses we determined the phosphorylation status of mitogen-activated protein kinase (MAPK) members. Incubation of cells with sildenafil increased both extracellular signal regulated kinase 1/2 (ERK1/2) and p38 phosphorylation in a time-dependent manner. Inhibition of MEK by PD98059 and p38 with SB203580 blocked sildenafil-induced proliferation and migration, respectively, suggesting that these MAPK members are downstream of PDE5 and mediate the angiogenic effects of sildenafil. PDE5 inhibitors could, thus, be used in disease states where neo-vessel growth is desired.     

Unc119 regulates myofibroblast differentiation through the activation of Fyn and the p38 MAPK pathway

Unc119 is an adaptor protein that is involved in the development of the vertebrate nervous system. We have shown that Unc119 stimulates the induction of alpha-smooth muscle actin (alpha-SMA) and myofibroblast differentiation by TGF-beta in human lung fibroblasts. Unc119 increases the kinase activity of Fyn and associates with it in coprecipitation and colocalization studies. Phosphorylation and activation of Fyn in response to TGF-beta and platelet-derived growth factor is delayed in Unc119-deficient cells. This delay translates into suppressed cell proliferation. In Src family kinase-deficient (SYF) cells, Unc119 knockdown does not affect cell proliferation. The result suggests that Unc119 interacts with Fyn in the early stages of signal generation and its presence is essential for conducive signal transduction. Unc119 overexpression does not stimulate alpha-SMA in SYF cells and this defect is restored upon reconstitution with Fyn indicating that Unc119 stimulation of alpha-SMA requires at least Fyn. Unc119 overexpression stimulated p38, but not JNK, phosphorylation. Blocking p38 MAPK resulted in reduced alpha-SMA expression by Unc119 suggesting that the p38 pathway regulates Unc119-induced myofibroblast differentiation. Unc119 stimulates the production of TGF-beta and IL-6, known inducers of myofibroblast differentiation. Thus, Unc119 regulates receptor-mediated signal transduction and myofibroblast differentiation by activating Fyn and the p38 MAPK pathway. Using primary lung fibroblasts from patients with fibrotic lung diseases and control subjects, we show that the expression of alpha-smooth muscle actin is highly correlated with that of Unc119. Taken together, our results suggest that Unc119 plays an important role in fibrotic processes through myofibroblast differentiation.     

Interaction of the SH2 domain of Fyn with a cytoskeletal protein, beta-adducin

Fyn is a Src family tyrosine kinase expressed abundantly in neurons and believed to have specific functions in the brain. To understand the function of Fyn tyrosine kinase, we attempted to identify Fyn Src homology 2 (SH2) domain-binding proteins from a Nonidet P-40-insoluble fraction of the mouse brain. beta-Adducin, an actin filament-associated cytoskeletal protein, was isolated by two-dimensional gel electrophoresis and identified by tandem mass spectrometry. beta-Adducin was tyrosine phosphorylated by coexpression with wild type but not with a kinase-negative form of Fyn in COS-7 cells. Cell staining analysis showed that coexpression of beta-adducin with Fyn induced translocation of beta-adducin from the cytoplasm to the periphery of the cells where it was colocalized with actin filaments and Fyn. These findings suggest that tyrosine-phosphorylated beta-adducin associates with the SH2 domain of Fyn and colocalizes under plasma membranes.     

c-Src-mediated phosphorylation of AP-2 reveals a general mechanism for receptors internalizing through the clathrin pathway

Clathrin-mediated endocytosis is a complex process regulated at many different levels. We showed previously that activation of the angiotensin type 1 receptor (AT1R), which belongs to the G protein-coupled receptor (GPCR) family, leads to c-Src-dependent tyrosine phosphorylation of beta2-adaptin, a subunit of the clathrin adaptor AP-2. The phosphorylation of beta2-adaptin on tyrosine residue 737 (Y737) negatively regulates its interaction with betaarrestin, another important clathrin adaptor for GPCR internalization. Here we sought to determine whether AP-2 phosphorylation represents a general mechanism for different receptors internalizing through the clathrin pathway. Using a specifically designed antibody against the phosphorylated form of Y737 on beta2-adaptin, we demonstrate that this residue is phosphorylated by AT1R in different cell types like HEK293, COS-7 and vascular smooth muscle cells. Using RNA interference approaches, we reveal that this agonist-mediated event is both betaarrestin- and c-Src-dependent, and that it occurs at the plasma membrane in clathrin-coated vesicles (CCVs). We further show that this is not only a common event employed by other GPCRs like the beta2-adrenergic, vasopressin V2, bradykinin type 2, platelet-activating factor and endothelin A receptors but that the epidermal growth factor receptor is capable of eliciting the phosphorylation of AP-2 in CCVs. Our results imply that tyrosine phosphorylation of Y737 on beta2-adaptin is a common regulatory mechanism employed by different receptors undergoing clathrin-dependent endocytosis, and suggest a wider function for this event than originally anticipated.     

WNK4 inhibits NCC protein expression through MAPK ERK1/2 signaling pathway

WNK [with no lysine (K)] kinase is a subfamily of serine/threonine kinases. Mutations in two members of this family (WNK1 and WNK4) cause pseudohypoaldosteronism type II featuring hypertension, hyperkalemia, and metabolic acidosis. WNK1 and WNK4 were shown to regulate sodium chloride cotransporter (NCC) activity through phosphorylating SPAK and OSR1. Previous studies including ours have also shown that WNK4 inhibits NCC function and its protein expression. A recent study reported that a phorbol ester inhibits NCC function via activation of extracellular signal-regulated kinase (ERK) 1/2 kinase. In the current study, we investigated whether WNK4 affects NCC via the MAPK ERK1/2 signaling pathway. We found that WNK4 increased ERK1/2 phosphorylation in a dose-dependent manner in mouse distal convoluted tubule (mDCT) cells, whereas WNK4 mutants with the PHA II mutations (E562K and R1185C) lost the ability to increase the ERK1/2 phosphorylation. Hypertonicity significantly increased ERK1/2 phosphorylation in mDCT cells. Knock-down of WNK4 expression by siRNA resulted in a decrease of ERK1/2 phosphorylation. We further showed that WNK4 knock-down significantly increases the cell surface and total NCC protein expressions and ERK1/2 knock-down also significantly increases cell surface and total NCC expression. These data suggest that WNK4 inhibits NCC through activating the MAPK ERK1/2 signaling pathway.     

Ligand binding up-regulates EphA2 messenger RNA through the mitogen-activated protein/extracellular signal-regulated kinase pathway

The EphA2 receptor tyrosine kinase is overexpressed in aggressive cancer cells, where it critically influences many aspects of malignant character. Although high levels of EphA2 have been documented in many different cancers, relatively little is known of the mechanisms that govern EphA2 gene expression in normal or malignant cells. Our present studies demonstrate that EphA2 influences the regulation of its own gene expression. Specifically, ligand-mediated phosphorylation of EphA2 transmits signals to the nucleus via extracellular signal-regulated kinase kinases to up-regulate de novo EphA2 gene expression and synthesis. This mechanism governs EphA2 expression in normal and malignant cells. In normal cells, EphA2 protein expression is balanced by ligand-mediated induction of EphA2 gene expression countered by EphA2 protein turnover. These findings suggest that EphA2 expression and ligand binding are intimately linked in epithelial cells. Increased understanding of this mechanism could have important implications for understanding the causes of EphA2 overexpression and for developing new strategies for therapeutic intervention in the many cancers that overexpress EphA2.     

The Polyhomeotic protein induces hyperplastic tissue overgrowth through the activation of the JAK/STAT pathway

Epigenetic mechanisms controlling cellular proliferation are essential to animal development. Moreover, altered levels of expression of the epigenetic regulator proteins are associated with the development and progression of human diseases like cancer. We have studied the effects of high levels of Polyhomeotic (PH) protein, a member of the Polycomb Group (PcG), during the proliferation of the imaginal discs in Drosophila. Over expression of PH protein causes induction of proliferation, accompanied with induction of JNK-dependent apoptosis. As a result, massive hyperplastic overgrowth is produced and the corresponding differentiated tissues show phenotypes related with mis-regulation of homeotic gene expression. We have found that high levels of PH up-regulate the JAK/STAT pathway through the de-repression of Unpaired (UPD), the extracellular ligand of the Drosophila JAK/STAT signalling cascade. Moreover, inactivation of the JAK/STAT pathway in the presence of a large amount of PH protein greatly reduces the tissue overgrowth, demonstrating a functional role of JAK/STAT in PH-induced hyperplasia. Finally, we have observed that decapentaplegic and d-myc, two growth genes and putative targets of the JAK/STAT pathway, are also over expressed in the PH-induced tumors. We propose that during normal development, the PcG proteins act to maintain inactive the JAK/STAT pathway. Upon cellular stress, changes in the levels of PcG proteins expression are induced and JAK/STAT is activated leading to tumor development. Our results show a functional relationship between the PcG gene expression and the JAK/STAT pathway, both of which are found to be perturbed in tumorigenesis.     

The sphingolipid long-chain base-Pkh1/2-Ypk1/2 signaling pathway regulates eisosome assembly and turnover

Eisosomes are recently described fungal structures that play roles in the organization of the plasma membrane and endocytosis. Their major protein components are Pil1 and Lsp1, and previous studies showed that these proteins are phosphorylated by the sphingolipid long-chain base-activated Pkh1 and Pkh2 protein kinases in vitro. We show that Pkh1 and Pkh2 phosphorylate Pil1 and Lsp1 in vivo to produce species B, and that heat stress, which activates Pkh1 and Pkh2, generates a more highly phosphorylated species, C. Cells with low Pkh activity lack species B and C and contain abnormally organized eisosomes. To verify that Pil1 phosphorylation is essential for correct eisosome organization, phosphorylated serine and threonine residues were identified and changed to alanines. A variant Pil1 protein lacking five phosphorylation sites did not form eisosomes during log phase growth, indicating that phosphorylation is critical for eisosome organization. We also found that eisosomes are dynamic structures and disassemble when the Ypk protein kinases, which are activated by the sphingolipid-Pkh signaling pathway, are inactivated or when the sphingolipid signal is pharmacologically blocked with myriocin. We conclude that eisosome formation and turnover are regulated by the sphingolipid-Pkh1/2-Ypk1/2 signaling pathway. These data and previous data showing that endocytosis is regulated by the sphingolipid-Pkh1/2-Ypk1/2 signaling pathway suggest that Pkh1 and -2 respond to changes in membrane sphingolipids and transmit this information to eisosomes via Pil1 phosphorylation. Eisosomes then control endocytosis to align the composition and function of the plasma membrane to match demand.     

Sendai virus assembly: M protein binds to viral glycoproteins in transit through the secretory pathway.

We have examined the relative ability of Sendai virus M (matrix) protein to associate with membranes containing viral glycoproteins at three distinct stages of the exocytic pathway prior to cell surface appearance. By the use of selective low-temperature incubations or the ionophore monensin, the transport of newly synthesized viral glycoproteins was restricted to either the pre-Golgi intermediate compartment (by incubation at 15 degrees C), the medial Golgi (in the presence of monensin), or the trans-Golgi network (by incubation at 20 degrees C). All three of these treatments resulted in a marked accumulation of the M protein on perinuclear Golgi-like membranes which in each case directly reflected the distribution of the viral F protein. Subsequent redistribution of the F protein to the plasma membrane by removal of the low-temperature (20 degrees C) block resulted in a concomitant redistribution of the M protein, thus implying association of the two components during intracellular transit. The extent of M protein-glycoprotein association was further examined by cell fractionation studies performed under each of the three restrictive conditions. Following equilibrium sedimentation of membranes derived from monensin-treated cells, approximately 40% of the recovered M protein was found to cofractionate with membranes containing the viral glycoproteins. Also, by flotation analyses, a comparable subpopulation of M protein was found to be membrane associated whether viral glycoproteins were restricted to the trans-Golgi network, the medial Golgi, or the pre-Golgi intermediate compartment. Additionally, transient expression of M protein alone from cloned cDNA showed that neither membrane association nor Golgi localization occurs in the absence of Sendai virus glycoproteins.     

The assembly pathway of outer membrane protein PhoE of Escherichia coli.

The assembly of the wild-type and several mutant forms of the trimeric outer membrane porin PhoE of Escherichia coli was investigated in vitro and in vivo. In in vivo pulse-chase experiments, approximately half of the wild-type PhoE molecules assembled within the 30-s pulse in the native conformation in the cell envelope. The other half of the molecules followed slower kinetics, and three intermediates in this multistep assembly process were detected: a soluble trypsin-sensitive monomer, a trypsin-sensitive monomeric form that was loosely associated with the cell envelope and a metastable trimer, which was integrated into the membranes and converted to the stable trimeric configuration within minutes. The metastable trimers disassembled during sample preparation for standard SDS/PAGE into folded monomers. In vitro, the isolated PhoE protein could efficiently be folded in the presence of N,N-dimethyldodecylamine-N-oxide (LDAO). A mutant PhoE protein, DeltaF330, which lacks the C-terminal phenylalanine residue, mainly followed the slower kinetic pathway observed in vivo, resulting in increased amounts of the various assembly intermediates. It appears that the DeltaF330 mutant protein is intrinsically able to fold, because it was able to fold in vitro with LDAO with similar efficiencies as the wild-type protein. Therefore, we propose that the conserved C-terminal Phe is (part of) a sorting signal, directing the protein efficiently to the outer membrane. Furthermore, we analysed a mutant protein with a hydrophilic residue introduced at the hydrophobic side of one of the membrane-spanning amphipathic beta strands. The assembly of this mutant protein was not affected in vivo or in vitro in the presence of LDAO. However, it was not able to form folded monomers in a previously established in vitro folding system, which requires the presence of lipopolysaccharides and Triton. Hence, a folded monomer might not be a true assembly intermediate of PhoE in vivo.