Effect of protein kinase A activity on the association of ADP-ribosylation factor 1 to Golgi membranes

The small GTP-binding protein ADP-ribosylation factor 1 (ARF1) is an essential component of the molecular machinery that catalyzes the formation of membrane-bound transport intermediates. By using an in vitro assay that reproduces recruitment of cytosolic proteins onto purified, high salt-washed Golgi membranes, we have analyzed the role of cAMP-dependent protein kinase A (PKA) on ARF1 incorporation. Addition to this assay of either pure catalytic subunits of PKA (C-PKA) or cAMP increased ARF1 binding. By contrast, ARF1 association was inhibited following C-PKA inactivation with either PKA inhibitory peptide or RIIalpha as well as after cytosol depletion of C-PKA. C-PKA also stimulated recruitment and activation of a recombinant form of human ARF1 in the absence of additional cytosolic components. The binding step could be dissociated from the activation reaction and found to be independent of guanine nucleotides and saturable. This step was stimulated by C-PKA in an ATP-dependent manner. Dephosphorylated Golgi membranes exhibited a decreased ability to recruit ARF1, and this effect was reverted by addition of C-PKA. Following an increase in the intracellular level of cAMP, ARF proteins redistributed from cytosol to the perinuclear Golgi region of intact cells. Collectively, the results show that PKA exerts a key regulatory role in the recruitment of ARF1 onto Golgi membranes. In contrast, PKA modulators did not affect recruitment of beta-COP onto Golgi membranes containing prebound ARF1

Secretory Component Delays the Conversion of Secretory IgA into Antigen-Binding Competent F(ab′)2: A Possible Implication for Mucosal Defense

AbstractSecretory component (SC) represents the soluble ectodomain of the polymeric Ig receptor, a membrane protein that transports mucosal Abs across epithelial cells. In the protease-rich environment of the intestine, SC is thought to stabilize the associated IgA by unestablished molecular mechanisms. To address this question, we reconstituted SC-IgA complexes in vitro by incubating dimeric IgA (IgAd) with either recombinant human SC (rSC) or SC isolated from human colostral milk (SCm). Both complexes exhibited an identical degree of covalency when exposed to redox agents, peptidyl disulfide isomerase, and temperature changes. In cross-competition experiments, 50% inhibition of binding to IgAd was achieved at ∼10 nM SC competitor. Western blot analysis of IgAd digested with intestinal washes indicated that the α-chain in IgAd was primarily split into a 40-kDa species, a phenomenon delayed in rSC- or SCm-IgAd complexes. In the same assay, either of the SCs was resistant to degradation only if complexed with IgAd. In contrast, the κ light chain was not digested at all, suggesting that the F(ab′)2 region was left intact. Accordingly, IgAd and SC-IgAd digestion products retained functionality as indicated by Ag reactivity in ELISA. Size exclusion chromatography under native conditions of digested IgAd and rSC-IgAd demonstrates that SC exerts its protective role in secretory IgA by delaying cleavage in the hinge/Fc region of the α-chain, not by holding together degraded fragments. The function of integral secretory IgA and F(ab′)2 is discussed in terms of mucosal immune defenses.</jats:p

In vivo kinetics of protein targeting to the endoplasmic reticulum determined by site-specific phosphorylation

We have developed a novel assay to detect the cytosolic localization of protein domains by inserting a short consensus sequence for phosphorylation by protein kinase A. In transfected COS-1 cells, this sequence was labeled efficiently with [(32)P]phosphate only when exposed to the cytosol and not when translocated into the lumen of the endoplasmic reticulum. The phosphorylation state of this sequence can therefore be used to determine the topology of membrane proteins. This assay is sufficiently sensitive to detect even the transient cytosolic exposure of the N-terminal domain of a membrane protein with a reverse signal-anchor sequence. The extent of phosphorylation per newly synthesized polypeptide was shown to reflect the time of exposure to the cytosol, which depends on translation, targeting and translocation of the N-terminus. By altering the length of the N-terminal domain or manipulating the translation rate, it was determined that protein targeting is rapid and requires only a few seconds. The rate of N-terminal translocation was estimated to be ∼1.6 times the rate of translation

Recruitment of coat proteins to liposomes and peptidoliposomes

Intracellular transport within the cell is generally mediated by membrane vesicles. Their formation is typically initiated by activation of small GTPases that then recruit cytosolic proteins to the membrane surface to form a coat, interact with cargo and accessory proteins, and deform the lipid bilayer to produce a transport vesicle. Liposomes proved to be a useful tool to study the molecular mechanisms of these processes in vitro. Here we describe the use of liposomes and peptidoliposomes presenting lipid-coupled cytosolic tails of cargo proteins for the in vitro analysis of the membrane recruitment of AP-1 adaptors in the process of forming AP-1/clathrin coats. AP-1 recruitment is mediated by the GTPase Arf1 and requires specific lipids and cargo signals. Interaction with cargo induces AP-1 oligomerization already in the absence of clathrin. Without cargo peptides, accessory proteins, such as amphiphysin 2, can be identified that stabilize AP-1 binding to liposomal membranes

ARF1.GTP, tyrosine-based signals, and phosphatidylinositol 4,5-bisphosphate constitute a minimal machinery to recruit the AP-1 clathrin adaptor to membranes

At the trans-Golgi network, clathrin coats containing AP-1 adaptor complexes are formed in an ARF1-dependent manner, generating vesicles transporting cargo proteins to endosomes. The mechanism of site-specific targeting of AP-1 and the role of cargo are poorly understood. We have developed an in vitro assay to study the recruitment of purified AP-1 adaptors to chemically defined liposomes presenting peptides corresponding to tyrosine-based sorting motifs. AP-1 recruitment was found to be dependent on myristoylated ARF1, GTP or nonhydrolyzable GTP-analogs, tyrosine signals, and small amounts of phosphoinositides, most prominently phosphatidylinositol 4,5-bisphosphate, in the absence of any additional cytosolic or membrane bound proteins. AP-1 from cytosol could be recruited to a tyrosine signal independently of the lipid composition, but the rate of recruitment was increased by phosphatidylinositol 4,5-bisphosphate. The results thus indicate that cargo proteins are involved in coat recruitment and that the local lipid composition contributes to specifying the site of vesicle formation

In Vitro Formation of Recycling Vesicles from Endosomes Requires Adaptor Protein-1/Clathrin and Is Regulated by Rab4 and the Connector Rabaptin-5

The involvement of clathrin and associated adaptor proteins in receptor recycling from endosomes back to the plasma membrane is controversial. We have used an in vitro assay to identify the molecular requirements for the formation of recycling vesicles. Cells expressing the asialoglycoprotein receptor H1, a typical recycling receptor, were surface biotinylated and then allowed to endocytose for 10 min. After stripping away surface-biotin, the cells were permeabilized and the cytosol washed away. In a temperature-, cytosol-, and nucleotide-dependent manner, the formation of sealed vesicles containing biotinylated H1 could be reconstituted. Vesicle formation was strongly inhibited upon immunodepletion of adaptor protein (AP)-1, but not of AP-2 or AP-3, from the cytosol, and was restored by readdition of purified AP-1. Vesicle formation was stimulated by supplemented clathrin, but inhibited by brefeldin A, consistent with the involvement of ARF1 and a brefeldin-sensitive guanine nucleotide exchange factor. The GTPase rab4, but not rab5, was required to generate endosome-derived vesicles. Depletion of rabaptin-5/rabex-5, a known interactor of both rab4 and γ-adaptin, stimulated and addition of the purified protein strongly inhibited vesicle production. The results indicate that recycling is mediated by AP-1/clathrin-coated vesicles and regulated by rab4 and rabaptin-5/rabex-5

Oligomerization and Dissociation of AP-1 Adaptors Are Regulated by Cargo Signals and by ArfGAP1-induced GTP Hydrolysis

The mechanism of AP-1/clathrin coat formation was analyzed using purified adaptor proteins and synthetic liposomes presenting tyrosine sorting signals. AP-1 adaptors recruited in the presence of Arf1·GTP and sorting signals were found to oligomerize to high-molecular-weight complexes even in the absence of clathrin. The appendage domains of the AP-1 adaptins were not required for oligomerization. On GTP hydrolysis induced by the GTPase-activating protein ArfGAP1, the complexes were disassembled and AP-1 dissociated from the membrane. AP-1 stimulated ArfGAP1 activity, suggesting a role of AP-1 in the regulation of the Arf1 “GTPase timer.” In the presence of cytosol, AP-1 could be recruited to liposomes without sorting signals, consistent with the existence of docking factors in the cytosol. Under these conditions, however, AP-1 remained monomeric, and recruitment in the presence of GTP was short-lived. Sorting signals allowed stable recruitment and oligomerization also in the presence of cytosol. These results suggest a mechanism whereby initial assembly of AP-1 with Arf1·GTP and ArfGAP1 on the membrane stimulates Arf1 GTPase activity, whereas interaction with cargo induces oligomerization and reduces the rate of GTP hydrolysis, thus contributing to efficient cargo sorting