Reversible phosphorylation--dephosphorylation determines the localization of rab4 during the cell cycle.

The ras-like GTP binding protein rab4 is the only known rab protein on endosomes that is phosphorylated during mitosis. Since a large fraction of rab4 accumulates in the cytosol in mitotic cells, we investigated the molecular mechanism controlling membrane association of rab4. We first show that human rab4 is phosphorylated by recombinant mammalian p34cdc2 kinase in vitro. Next, the actual site of phosphorylation and its functional significance were determined using stably transfected CHO cell lines producing high levels of wild type rab4 or rab4 mutants bearing alterations at Ser196, which occurs within a consensus site for p34cdc2 kinase phosphorylation (S196PRR). Mutation of Ser196 to glutamine or aspartic acid completely prevented rab4 phosphorylation in mitotic cells and also blocked its appearance in the cytosol. Neither C-terminal isoprenylation nor carboxymethylation of rab4 was affected by the mutations or by phosphorylation. Finally, dephosphorylation and reassociation of soluble rab4 with membranes occurred upon exit of cells from mitosis. Thus, phosphorylation of Ser196 is directly responsible for the reversible translocation of rab4 into the cytosol of mitotic cells.     

Rab4 function in membrane recycling from early endosomes depends on a membrane to cytoplasm cycle

The monomeric GTPase rab4 is associated with early endosomes and regulates recycling vesicle formation. Because the function of rab proteins in the biosynthetic pathway does not appear to depend on cycling between membranes and cytosol, we were interested to investigate whether or not this holds true for rab function in the endocytic pathway. We created a chimeric rab4 protein (NHrab4cbvn) in which the carboxyl-terminal prenylation motif was replaced by the transmembrane domain of cellubrevin. The chimeric protein was permanently attached to membranes, properly targeted to early endosomes, and bound guanine nucleotide to the same extent as wild type rab4. However, in transport assays we found that basolaterally endocytosed transferrin was less efficiently transported to the apical cell surface in Madin-Darby canine kidney cells transfected with NHrab4cbvn than in cells expressing wild type rab4. Hence, rab4 function requires ongoing cycles of association and dissociation from early endosomes. This cycle is altered during mitosis when rab4 accumulates in the cytoplasm through phosphorylation by a mitotic kinase. We show here, using a rab4 construct that is permanently hooked onto membranes, that the membrane-bound pool of rab4 is targeted by a mitotic kinase.     

Binding of Rab3A to synaptic vesicles

Prenylated Rab GTPases cycle between membrane-bound and soluble forms. Membrane-bound GDP-Rabs interact with GDP dissociation inhibitor (GDI), resulting in the dissociation of a Rab.GDI complex, which in turn serves as a precursor for the membrane re-association of Rabs. We have now characterized the binding of Rab3A to synaptic vesicles in vitro using either purified complexes or rat brain cytosol as source for GDI.Rab3A. Binding of Rab3A results in the immediate release of GDI from the membrane. Furthermore, binding does not require the presence of additional guanine nucleotides (GDP or GTP) or of cytosolic factors. Although nucleotide exchange follows binding, binding is initially reversible, suggesting that binding of GDP-Rab3A and nucleotide exchange are separate and independent events. Comparison with the binding of Rab1B revealed that both Rab proteins bind preferentially to their respective resident membranes although some promiscuity was observable. Binding is saturable and involves a protease-sensitive binding site that is tightly associated with the vesicle membrane.     

Nuclear envelope precursor vesicle targeting to chromatin is stimulated by protein phosphatase 1 in Xenopus egg extracts

The mechanism underlying targeting of the nuclear membrane to chromatin at the end of mitosis was studied using an in vitro cell-free system comprising Xenopus egg membrane and cytosol fractions, and sperm chromatin. The mitotic phase membrane, which was separated from a mitotic phase extract of Xenopus eggs and could not bind to chromatin, became able to bind to chromatin on pretreatment with a synthetic phase cytosol fraction of Xenopus eggs. When the cytosol fraction was depleted of protein phosphatase 1 (PP1) with anti-Xenopus PP1γ1 antibodies, this ability was lost. The addition of recombinant xPP1γ1 to the PP1-depleted cytosol fraction restored the ability. These and other results suggested that dephosphorylation of mitotic phosphorylation sites on membranes by PP1 in the synthetic phase cytosol fraction promoted targeting of the membranes to chromatin. On the other hand, a fragment containing the chromatin-binding domain of lamin B receptor (LBR) but not emerin inhibited targeting of membrane vesicles. It was also shown that PP1 dephosphorylates a phosphate group(s) responsible for regulation of the binding of LBR to chromatin. A possible mechanism involving PP1 and LBR for the regulation of nuclear membrane targeting to chromatin was discussed.     

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 gamma-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.     

GDI1 encodes a GDP dissociation inhibitor that plays an essential role in the yeast secretory pathway.

GTP binding proteins of the Sec4/Ypt/rab family regulate distinct vesicular traffic events in eukaryotic cells. We have cloned GDI1, an essential homolog of bovine rab GDI (GDP dissociation inhibitor) from the yeast Saccharomyces cerevisiae. Analogous to the bovine protein, purified Gdi1p slows the dissociation of GDP from Sec4p and releases the GDP-bound form from yeast membranes. Depletion of Gdi1p in vivo leads to loss of the soluble pool of Sec4p and inhibition of protein transport at multiple stages of the secretory pathway. Complementation analysis indicates that GDI1 is allelic to sec19-1. These results establish that Gdi1p plays an essential function in membrane traffic and are consistent with a role for Gdi1p in the recycling of proteins of the Sec4/Ypt/rab family from their target membranes back to their vesicular pools.     

Accumulation of rab4GTP in the cytoplasm and association with the peptidyl-prolyl isomerase pin1 during mitosis

Transport through the endocytic pathway is inhibited during mitosis. The mechanism responsible for this inhibition is not understood. Rab4 might be one of the proteins involved as it regulates transport through early endosomes, is phosphorylated by p34(cdc2) kinase, and is translocated from early endosomes to the cytoplasm during mitosis. We investigated the perturbation of the rab4 GTPase cycle during mitosis. Newly synthesized rab4 was less efficiently targeted to membranes during mitosis. By subcellular fractionation of mitotic cells, we found a large increase of cytosolic rab4 in the active GTP-form, an increase not associated with the cytosolic rabGDP chaperone GDI. Instead, phosphorylated rab4 is in a complex with the peptidyl-prolyl isomerase Pin1 during mitosis, but not during interphase. Our results show that less efficient recruitment of rab4 to membranes and a bypass of the normal GDI-mediated retrieval of rab4GDP from early endosomes reduce the amount of rab4GTP on membranes during mitosis. We propose that phosphorylation of rab4 inhibits both the recruitment of rab4 effector proteins to early endosomes and the docking of rab4-containing transport vesicles. This mechanism might contribute to the inhibition of endocytic membrane transport during mitosis.     

Rab escort protein-1 is a multifunctional protein that accompanies newly prenylated rab proteins to their target membranes.

Rab proteins comprise a family of small GTPases that serve a regulatory role in vesicular membrane traffic. Geranylgeranylation of these proteins on C-terminal cysteine motifs is crucial for their membrane association and function. This post-translational modification is catalysed by rab geranylgeranyl transferase (Rab-GGTase), a multisubunit enzyme consisting of a catalytic heterodimer and an accessory component, named rab escort protein (REP)-1. Previous in vitro studies have suggested that REP-1 presents newly synthesized rab proteins to the catalytic component of the enzyme, and forms a stable complex with the prenylated proteins following the transfer reaction. According to this model, a cellular factor would be required to dissociate the rab protein from REP-1 and to allow it to recycle in the prenylation reaction. RabGDP dissociation inhibitor (RabGDI) was considered an ideal candidate for this role, given its established function in mediating membrane association of prenylated rab proteins. Here we demonstrate that dissociation from REP-1 and binding of rab proteins to the membrane do not require RabGDI or other cytosolic factors. The mechanism of REP-1-mediated membrane association of rab5 appears to be very similar to that mediated by RabGDI. Furthermore, REP-1 and RabGDI share several other functional properties, the ability to inhibit the release of GDP and to remove rab proteins from membranes; however, RabGDI cannot assist in the prenylation reaction. These data suggest that REP-1 is per se sufficient to chaperone newly prenylated rab proteins to their target membranes.     

Association and Colocalization of Eps15 with Adaptor Protein-2 and Clathrin

Eps15 has been identified as a substrate of the EGF receptor tyrosine kinase. In this report, we show that activation of the EGF receptor by either EGF or TGF-α results in phosphorylation of Eps15. Stimulation of cells with PDGF or insulin did not lead to Eps15 phosphorylation, suggesting that phosphorylation of Eps15 is a receptor-specific process. We demonstrate that Eps15 is constitutively associated with both α-adaptin and clathrin. Upon EGF stimulation, Eps15 and α-adaptin are recruited to the EGF receptor. Using a truncated EGF receptor mutant, we demonstrate that the regulatory domain of the cytoplasmic tail of the EGF receptor is essential for the binding of Eps15. Fractionation studies reveal that Eps15 is present in cell fractions enriched for plasma membrane and endosomal membranes. Immunofluorescence studies show that Eps15 colocalizes with adaptor protein-2 (AP-2) and partially with clathrin. No colocalization of Eps15 was observed with the early endosomal markers rab4 and rab5. These observations indicate that Eps15 is present in coated pits and coated vesicles of the clathrin-mediated endocytic pathway, but not in early endosomes. Neither AP-2 nor clathrin are required for the binding of Eps15 to coated pits or coated vesicles, since in membranes lacking AP-2 and clathrin, Eps15 still shows the same staining pattern. These findings suggest that Eps15 may play a critical role in the recruitment of active EGF receptors into coated pit regions before endocytosis of ligand-occupied EGF receptors.     

Thyrotropin (TSH) binding activities in bovine thyroid tissue: possible role of adenosine 3',5'-monophosphate dependent phosphorylation of thyroid plasma membranes in TSH receptor degradation

The relationship between thyroid plasma membrane phosphorylation and thyrotropin (TSH) receptor degradation was investigated by using bovine thyroid tissues. By fractionation of thyroid cytosol (105,000 X g supernatant of thyroid homogenate) in a continuous sucrose density gradient centrifugation, three different TSH binding activities were separated. During the incubation of thyroid plasma membranes, TSH binding activities were spontaneously released in vitro. By fractionation of the fraction containing released TSH binding activities in the same sucrose density gradient centrifugation, three different TSH binding activities were isolated. These peaks of TSH binding activity corresponded to the peaks of TSH binding activity obtained in cytosol fraction. Adenosine 3',5'-monophosphate (cyclic AMP) enhanced the release of TSH binding activities from the plasma membranes in vitro. After fractionation on a sucrose density gradient centrifugation of the supernatant of the plasma membranes which were preincubated with cyclic AMP, three different peaks of TSH binding activity were identified. These peaks corresponded to the peaks obtained in spontaneously released TSH binding activity. In this case, however, the amount of small molecule TSH binding activities was predominant compared to that of large molecule TSH binding activity. During the incubation of the plasma membranes with [r-32P]-ATP and with cyclic AMP, phosphorylated soluble proteins were released. The profile of the phosphorylated soluble proteins in the sucrose density gradient centrifugation showed three different peaks which corresponded to the peaks of binding activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

GTP hydrolysis is required for vesicle fusion during nuclear envelope assembly in vitro

Nuclear envelope assembly was studied in vitro using extracts from Xenopus eggs. Nuclear-specific vesicles bound to demembranated sperm chromatin but did not fuse in the absence of cytosol. Addition of cytosol stimulated vesicle fusion, pore complex assembly, and eventual nuclear envelope growth. Vesicle binding and fusion were assayed by light and electron microscopy. Addition of ATP and GTP to bound vesicles caused limited vesicle fusion, but enclosure of the chromatin was not observed. This result suggested that nondialyzable soluble components were required for nuclear vesicle fusion. GTP gamma S and guanylyl imidodiphosphate significantly inhibited vesicle fusion but had no effect on vesicle binding to chromatin. Preincubation of membranes with 1 mM GTP gamma S or GTP did not impair vesicle binding or fusion when assayed with fresh cytosol. However, preincubation of membranes with GTP gamma S plus cytosol caused irreversible inhibition of fusion. The soluble factor mediating the inhibition by GTP gamma S, which we named GTP-dependent soluble factor (GSF), was titratable and was depleted from cytosol by incubation with excess membranes plus GTP gamma S, suggesting a stoichiometric interaction between GSF and a membrane component in the presence of GTP gamma S. In preliminary experiments, cytosol depleted of GSF remained active for fusion of chromatin-bound vesicles, suggesting that GSF may not be required for the fusion reaction itself. We propose that GTP hydrolysis is required at a step before the fusion of nuclear vesicles.     

Soluble precursor of membrane-associated protein kinase in uterine smooth muscle cells

Incubation of smooth muscle strips from rat uterus with isoproterenol resulted in redistribution of protein kinase activity between the cytosol and a 20,000 to 50,000g membrane fraction. Similarities in the elution properties of the cytosolic and membrane-associated forms of the enzyme on DEAE-cellulose ion exchange chromatography further suggested the two forms were the same. The nature of membrane binding of the soluble enzyme was investigated using smooth muscle microsomal and cytosol fractions. Membranes readily bound the soluble enzyme when the two subcellular compartments were reconstituted and incubated at 30 °C for 10 min. The extent of binding was proportional to the ratio of membranes to cytosol and was characterized by the inhibition of soluble enzyme activity toward exogenous substrates in a Triton X-100 reversible manner. In marked contrast to the binding of soluble protein kinase to heart particulate fractions, binding of the cytosol enzyme to smooth muscle cell membranes was unaffected by ionic strength or cAMP. The latter property indicated holoenzyme was bound in a manner similar to the free catalytic subunit of cAMP-dependent protein kinase and suggested the enzyme was bound by association between the membrane and the catalytic subunit. Binding of cytosol protein kinase to the membranes rendered the enzyme insensitive to trypsin digestion and the capacity of the smooth muscle cell membranes to bind the soluble enzyme exceeded that of other rat tissue fractions. Resistance to salt extraction and proteolysis, as well as its detergent dependence, suggested the soluble enzyme became an integral or intrinsic membrane protein following association with the membrane. The ability of membranes to incorporate [γ-³²P]ATP into phosphoprotein was lost on detergent extraction of protein kinase and restored in an apparently specific manner when extracted and washed membranes were reconstituted with soluble enzyme. The intrinsic nature of membrane protein kinase and the apparent specificity with which the soluble enzyme was hound by membranes further indicated that, in myometrium. hormone-induced translocation of protein kinase is an important mechanism by which enzyme activity is increased in the vicinity of its in situ substrates.     

Active ADP-ribosylation factor-1 (ARF1) is required for mitotic Golgi fragmentation

In mammalian cells the Golgi apparatus undergoes an extensive disassembly process at the onset of mitosis that is believed to facilitate equal partitioning of this organelle into the two daughter cells. However, the underlying mechanisms for this fragmentation process are so far unclear. Here we have investigated the role of the ADP-ribosylation factor-1 (ARF1) in this process to determine whether Golgi fragmentation in mitosis is mediated by vesicle budding. ARF1 is a small GTPase that is required for COPI vesicle formation from the Golgi membranes. Treatment of Golgi membranes with mitotic cytosol or with purified coatomer together with wild type ARF1 or its constitutive active form, but not the inactive mutant, converted the Golgi membranes into COPI vesicles. ARF1-depleted mitotic cytosol failed to fragment Golgi membranes. ARF1 is associated with Golgi vesicles generated in vitro and with vesicles in mitotic cells. In addition, microinjection of constitutive active ARF1 did not affect mitotic Golgi fragmentation or cell progression through mitosis. Our results show that ARF1 is active during mitosis and that this activity is required for mitotic Golgi fragmentation.     

RhoGDIs revisited: novel roles in Rho regulation

Small GTP-binding proteins of the Rho/Rac/Cdc42 family combine their GDP/GTP cycle, regulated by guanine nucleotide-exchange factors and GTPase-activating proteins, to a cytosol/membrane cycle, regulated by guanine nucleotide dissociation inhibitors (rhoGDIs). RhoGDIs are endowed with dual functions in the cytosol where they form soluble complexes with geranylgeranylated GDP-bound Rho proteins and at membrane interfaces where they monitor the delivery and extraction of Rho proteins to/from their site of action. They have little diversity compared with other Rho protein regulators and therefore have been regarded mostly as housekeeping regulators that distribute Rho proteins equally to any membranes. Recently, acquired data show that rhoGDIs, by interacting with candidate receptors/displacement factors or by phosphorylation, may in fact have active contributions to targeting Rho proteins to specific subcellular membranes and signaling pathways. In addition, the GDP/GTP and membrane/cytosol cycles can be uncoupled in certain cases, with Rho proteins either escaping the membrane/cytosol cycle or being regulated by rhoGDIs in their GTP-bound form. Here, we survey recent structure-function relationships and cellular studies on rhoGDIs and revisit their classical housekeeping role into novel and more specific functions. We also review their involvement in diseases.     

Regulation of ARNO nucleotide exchange by a PH domain electrostatic switch.

ARNO is a member of a family of guanine nucleotide exchange factors that activate small GTPases called ADP-ribosylation factors (ARFs) [1] [2] [3], which regulate vesicular trafficking and, in one case (ARF6), also regulate cortical actin structure [4]. ARNO is located at the plasma membrane, and in the presence of activated protein kinase C (PKC) can induce cortical actin rearrangements reminiscent of those produced by active ARF6 [5] [6] [7] [8]. High-affinity binding of ARNO to membranes, which is required for exchange activity, is mediated cooperatively by a pleckstrin homology (PH) domain and an adjacent carboxy-terminal polybasic domain [3] [9]. ARNO is phosphorylated in vivo by PKC on a single serine residue, S392, located within the carboxy-terminal polybasic domain. Mutation of S392 to alanine does not prevent ARNO-mediated actin rearrangements, suggesting that phosphorylation does not lead to ARNO activation [6]. Here, we report that phosphorylation negatively regulates ARNO exchange activity through a 'PH domain electrostatic switch'. Introduction of a negatively charged phosphate into the polybasic domain reduced interaction of ARNO with membranes both in vitro and in vivo, and inhibited exchange in vitro. This regulated membrane association is similar to the myristoyl electrostatic switch that controls membrane binding of the myristoylated alanine-rich C kinase substrate (MARCKS) [10], but to our knowledge is the first demonstration of an electrostatic switch regulating the membrane interaction of a protein containing a PH domain. This mechanism allows regulation of ARNO lipid binding and exchange activity at two levels, phosphoinositide-dependent recruitment and PKC-dependent displacement from the membrane.     

Dissection of membrane dynamics of the ARF-guanine nucleotide exchange factor GBF1

ADP-ribosylation factor (ARF)-facilitated recruitment of COP I to membranes is required for secretory traffic. The guanine nucleotide exchange factor GBF1 activates ARF and regulates ARF/COP I dynamics at the endoplasmic reticulum (ER)-Golgi interface. Like ARF and coatomer, GBF1 peripherally associates with membranes. ADP-ribosylation factor and coatomer have been shown to rapidly cycle between membranes and cytosol, but the membrane dynamics of GBF1 are unknown. Here, we used fluorescence recovery after photobleaching to characterize the behavior of GFP-tagged GBF1. We report that GBF1 rapidly cycles between membranes and the cytosol (t1/2 is approximately 17 +/- 1 seconds). GBF1 cycles faster than GFP-tagged ARF, suggesting that in each round of association/dissociation, GBF1 catalyzes a single event of ARF activation, and that the activated ARF remains on membrane after GBF1 dissociation. Using three different approaches [expression of an inactive (E794K) GBF1 mutant, expression of the ARF1 (T31N) mutant with decreased affinity for GTP and Brefeldin A treatment], we show that GBF1 is stabilized on membranes when in a complex with ARF-GDP. GBF1 dissociation from ARF and membranes is triggered by its catalytic activity, i.e. the displacement of GDP and the subsequent binding of GTP to ARF. Our findings imply that continuous cycles of recruitment and dissociation of GBF1 to membranes are required for sustained ARF activation and COP I recruitment that underlies ER-Golgi traffic.     

The N-terminal domain of a rab protein is involved in membrane-membrane recognition and/or fusion.

Proteins of the YPT1/SEC4/rab family are well documented to be involved in the regulation of membrane transport. We have previously reported that rab5 regulates endosome-endosome recognition and/or fusion in vitro. Here, we show that this process depends on the rab5 N-terminal domain. Treatment of early endosomal membranes at a low trypsin concentration essentially abolished fusion and cleaved rab5 to a 1 kDa smaller polypeptide. Two-dimensional gel analysis suggested that rab5 is one of the few, if not the only, polypeptides cleaved by trypsin under these conditions. Whereas endosome fusion could be stimulated by cytosol prepared from cells overexpressing rab5 (and thus containing high amounts of the protein), this stimulation was abolished by trypsin-treatment of the cytosol. Trypsin-treated cytosol prepared from mock-transfected cells, which contains very low amounts of rab5, showed no inhibitory activity indicating that rab5 is the target of trypsin in these experiments. Purified rab5 prepared after expression in Escherichia coli was treated with trypsin, which cleaved the protein at the N-terminus. A synthetic peptide of rab5 N-terminal domain inhibited endosome fusion in our cell-free assay. A version of the same peptide truncated at the N-terminus or a peptide of rab3 N-terminal domain were without effects. Altogether, these observations suggest that the N-terminal domain of rab5 is involved in the process of early endosome recognition and/or fusion, presumably because it interacts with another component of the transport machinery.     

Subcellular distribution of alpha 2-adrenergic receptors, pertussis-toxin substrate and adenylate cyclase in human platelets.

The subcellular distribution of the alpha 2-adrenergic receptor, pertussis-toxin substrates (Gi, the inhibitory G-protein) and adenylate cyclase was determined in human platelets. The alpha 2-adrenergic receptor and pertussis-toxin substrate activity codistribute with surface membranes identified by a novel fluorescent-lectin method. The platelet granule fractions did not contain detectable Gi. Only 2-4% of the total pertussis-toxin substrate activity appears in soluble fractions, and this amount was not increased upon addition of purified beta gamma units or after pretreatment of platelets with adrenaline. There is no evidence for compartmentation of the alpha 2-adrenergic receptor or Gi to account for the low-affinity component of agonist binding to the alpha 2-adrenergic receptor in human platelet membranes. Translocation of Gi from plasma membrane to platelet cytosol or granules does not appear to play any significant role in the mechanism of alpha 2-receptor-mediated platelet activation.     

Catalytic properties of the Escherichia coli proton adenosinetriphosphatase: evidence that nucleotide bound at noncatalytic sites is not involved in regulation of oxidative phosphorylation

Nucleotide-depleted F1-ATPase from Escherichia coli was reconstituted with F1-depleted membranes and shown to catalyze high rates of oxidative phosphorylation of ADP and GDP. Adenine nucleotide became bound to the nonexchangeable nucleotide sites on membrane-bound F1 during ATP synthesis, but binding of guanine nucleotides to nonexchangeable sites during GTP synthesis was not detectable. It was possible to reload the nonexchangeable sites on nucleotide-depleted F1 with radioactive adenine nucleotide prior to membrane reconstitution. The radioactive adenine nucleotide did not exchange significantly during oxidative phosphorylation of ADP or GDP. The amount of nonexchangeable adenine nucleotide found in membrane-bound F1 was the same when the nonexchangeable sites were reloaded either prior to membrane reconstitution of the F1 or after membrane reconstitution with nucleotide-free F1 followed by a burst of oxidative phosphorylation of ADP. The results showed that occupation of the nonexchangeable sites on F1 by tightly bound nucleotide is not required for oxidative phosphorylation of GDP (a physiological activity of F1 in the bacterial cell). Also, the results confirm directly that the adenine-specific nonexchangeable sites on F1 are noncatalytic sites. Using this experimental approach, it was possible to look for a regulatory effect of the nonexchangeable nucleotide on oxidative phosphorylation. Nucleotide-depleted F1 was first reloaded with (i) ATP, (ii) ADP, (iii) 5'-adenylyl imidodiphosphate, or (iv) zero nucleotide, and was then reconstituted with F1-depleted membranes. The reconstituted membranes were compared in respect to rates of oxidative phosphorylation of GDP and Km values of GDP and Pi. No regulatory role for the nonexchangeable nucleotide was evident.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of protein phosphorylation in Dictyostelium discoideum

We have examined the phosphorylation of the cyclic adenosine 3':5' monophosphate (cAMP) cell surface chemotactic receptor and a 36 kDa membrane-associated protein (p36) in Dictyostelium discoideum. The activity of CAR-kinase, the enzyme responsible for the phosphorylation of the cAMP receptor, was studied in plasma membrane preparations. It was found that, as in intact cells, the receptor was rapidly phosphorylated in membranes incubated with [gamma 32P] adenosine triphosphate (ATP) but only in the presence of cAMP. This phosphorylation was not observed in membranes prepared from cells which did not display significant cAMP binding activity. cAMP could induce receptor phosphorylation at low concentrations, while cyclic guanosine 3':5' monophosphate (cGMP) could elicit receptor phosphorylation only at high concentrations. Neither ConA, Ca2+, or guanine nucleotides had an effect on CAR-kinase. It was also observed that 2-deoxy cAMP but not dibutyryl cAMP induced receptor phosphorylation. The data suggest that the ligand occupied form of the cAMP receptor is required for CAR-kinase activity. Although the receptor is rapidly dephosphorylated in vivo, we were unable to observe its dephosphorylation in vitro. In contrast, p36 was rapidly dephosphorylated. Also, unlike the cAMP receptor, the phosphorylation of p36 was found to be regulated by the addition of guanine nucleotides. Guanosine diphosphate (GDP) enhanced the phosphorylation while guanosine triphosphate (GTP) decreased the radiolabeling of p36 indicating that GTP can compete with ATP for the nucleotide triphosphate binding site of p36 kinase. Thus was verified using radiolabeled GTP as the phosphate donor. Competition experiments with GTP gamma S, ATP, GTP, CTP, and uridine triphosphate (UTP) indicated that the phosphate donor site of p36 kinase is relatively non-specific.(ABSTRACT TRUNCATED AT 250 WORDS)