Proton uptake by bacterial reaction centers: The protein complex responds in a similar manner to the reduction of either quinone acceptor

In bacterial photosynthetic reaction centers, the protonation events associated with the different reduction states of the two quinone molecules constitute intrinsic probes of both the electrostatic interactions and the different kinetic events occurring within the protein in response to the light-generated introduction of a charge. The kinetics and stoichiometries of proton uptake on formation of the primary semiquinone QA− and the secondary acceptor QB− after the first and second flashes have been measured, at pH 7.5, in reaction centers from genetically modified strains and from the wild type. The modified strains are mutated at the L212Glu and/or at the L213Asp sites near QB; some of them carry additional mutations distant from the quinone sites (M231Arg → Leu, M43Asn → Asp, M5Asn → Asp) that compensate for the loss of L213Asp. Our data show that the mutations perturb the response of the protein system to the formation of a semiquinone, how distant compensatory mutations can restore the normal response, and the activity of a tyrosine residue (M247Ala → Tyr) in increasing and accelerating proton uptake. The data demonstrate a direct correlation between the kinetic events of proton uptake that are observed with the formation of either QA− or QB−, suggesting that the same residues respond to the generation of either semiquinone species. Therefore, the efficiency of transferring the first proton to QB is evident from examination of the pattern of H+/QA− proton uptake. This delocalized response of the protein complex to the introduction of a charge is coordinated by an interactive network that links the Q− species, polarizable residues, and numerous water molecules that are located in this region of the reaction center structure. This could be a general property of transmembrane redox proteins that couple electron transfer to proton uptake/release reactions.

ADP-Ribosylation Factor 1 Transiently Activates High-Affinity Adaptor Protein Complex AP-1 Binding Sites On Golgi Membranes

Association of the Golgi-specific adaptor protein complex 1 (AP-1) with the membrane is a prerequisite for clathrin coat assembly on the trans-Golgi network (TGN). The AP-1 adaptor is efficiently recruited from cytosol onto the TGN by myristoylated ADP-ribosylation factor 1 (ARF1) in the presence of the poorly hydrolyzable GTP analog guanosine 5′-O-(3-thiotriphosphate) (GTPγS). Substituting GTP for GTPγS, however, results in only poor AP-1 binding. Here we show that both AP-1 and clathrin can be recruited efficiently onto the TGN in the presence of GTP when cytosol is supplemented with ARF1. Optimal recruitment occurs at 4 μM ARF1 and with 1 mM GTP. The AP-1 recruited by ARF1·GTP is released from the Golgi membrane by treatment with 1 M Tris-HCl (pH 7) or upon reincubation at 37°C, whereas AP-1 recruited with GTPγS or by a constitutively active point mutant, ARF1(Q71L), remains membrane bound after either treatment. An incubation performed with added ARF1, GTP, and AlFn, used to block ARF GTPase-activating protein activity, results in membrane-associated AP-1, which is largely insensitive to Tris extraction. Thus, ARF1·GTP hydrolysis results in lower-affinity binding of AP-1 to the TGN. Using two-stage assays in which ARF1·GTP first primes the Golgi membrane at 37°C, followed by AP-1 binding on ice, we find that the high-affinity nucleating sites generated in the priming stage are rapidly lost. In addition, the AP-1 bound to primed Golgi membranes during a second-stage incubation on ice is fully sensitive to Tris extraction, indicating that the priming stage has passed the ARF1·GTP hydrolysis point. Thus, hydrolysis of ARF1·GTP at the priming sites can occur even before AP-1 binding. Our finding that purified clathrin-coated vesicles contain little ARF1 supports the concept that ARF1 functions in the coat assembly process rather than during the vesicle-uncoating step. We conclude that ARF1 is a limiting factor in the GTP-stimulated recruitment of AP-1 in vitro and that it appears to function in a stoichiometric manner to generate high-affinity AP-1 binding sites that have a relatively short half-life.

A General RNA-Binding Protein Complex That Includes the Cytoskeleton-associated Protein MAP 1A

Association of mRNA with the cytoskeleton represents a fundamental aspect of RNA physiology likely involved in mRNA transport, anchoring, translation, and turnover. We report the initial characterization of a protein complex that binds RNA in a sequence-independent but size-dependent manner in vitro. The complex includes a ∼160-kDa protein that is bound directly to mRNA and that appears to be either identical or highly related to a ∼1600-kDa protein that binds directly to mRNA in vivo. In addition, the microtubule-associated protein, MAP 1A, a cytoskeletal associated protein is a component of this complex. We suggest that the general attachment of mRNA to the cytoskeleton may be mediated, in part, through the formation of this ribonucleoprotein complex.

Characterization of a Fourth Adaptor-related Protein Complex

Adaptor protein complexes (APs) function as vesicle coat components in different membrane traffic pathways; however, there are a number of pathways for which there is still no candidate coat. To find novel coat components related to AP complexes, we have searched the expressed sequence tag database and have identified, cloned, and sequenced a new member of each of the four AP subunit families. We have shown by a combination of coimmunoprecipitation and yeast two-hybrid analysis that these four proteins (ε, β4, μ4, and ς4) are components of a novel adaptor-like heterotetrameric complex, which we are calling AP-4. Immunofluorescence reveals that AP-4 is localized to ∼10–20 discrete dots in the perinuclear region of the cell. This pattern is disrupted by treating the cells with brefeldin A, indicating that, like other coat proteins, the association of AP-4 with membranes is regulated by the small GTPase ARF. Immunogold electron microscopy indicates that AP-4 is associated with nonclathrin-coated vesicles in the region of the trans-Golgi network. The μ4 subunit of the complex specifically interacts with a tyrosine-based sorting signal, indicating that, like the other three AP complexes, AP-4 is involved in the recognition and sorting of cargo proteins with tyrosine-based motifs. AP-4 is of relatively low abundance, but it is expressed ubiquitously, suggesting that it participates in a specialized trafficking pathway but one that is required in all cell types.

A Novel RING Finger Protein Complex Essential for a Late Step in Protein Transport to the Yeast Vacuole

Protein transport to the lysosome-like vacuole in yeast is mediated by multiple pathways, including the biosynthetic routes for vacuolar hydrolases, the endocytic pathway, and autophagy. Among the more than 40 genes required for vacuolar protein sorting (VPS) in Saccharomyces cerevisiae, mutations in the four class C VPS genes result in the most severe vacuolar protein sorting and morphology defects. Herein, we provide complementary genetic and biochemical evidence that the class C VPS gene products (Vps18p, Vps11p, Vps16p, and Vps33p) physically and functionally interact to mediate a late step in protein transport to the vacuole. Chemical cross-linking experiments demonstrated that Vps11p and Vps18p, which both contain RING finger zinc-binding domains, are components of a hetero-oligomeric protein complex that includes Vps16p and the Sec1p homologue Vps33p. The class C Vps protein complex colocalized with vacuolar membranes and a distinct dense membrane fraction. Analysis of cells harboring a temperature-conditional vps18 allele (vps18tsf) indicated that Vps18p function is required for the biosynthetic, endocytic, and autophagic protein transport pathways to the vacuole. In addition, vps18tsf cells accumulated multivesicular bodies, autophagosomes, and other membrane compartments that appear to represent blocked transport intermediates. Overproduction of either Vps16p or the vacuolar syntaxin homologue Vam3p suppressed defects associated with vps18tsf mutant cells, indicating that the class C Vps proteins and Vam3p may functionally interact. Thus we propose that the class C Vps proteins are components of a hetero-oligomeric protein complex that mediates the delivery of multiple transport intermediates to the vacuole.

Computer-based analysis of the binding steps in protein complex formation

Computer models were used to examine whether and under what conditions the multimeric protein complex is inhibited by high concentrations of one of its components—an effect analogous to the prozone phenomenon in precipitin tests. A series of idealized simple “ball-and-stick” structures representing small oligomeric complexes of protein molecules formed by reversible binding reactions were analyzed to determine the binding steps leading to each structure. The equilibrium state of each system was then determined over a range of starting concentrations and Kds and the steady-state concentration of structurally complete oligomer calculated for each situation. A strong inhibitory effect at high concentrations was shown by any protein molecule forming a bridge between two or more separable parts of the complex. By contrast, proteins linked to the outside of the complex by a single bond showed no inhibition whatsoever at any concentration. Nonbridging, multivalent proteins in the body of the complex could show an inhibitory effect or not depending on the structure of the complex and the strength of its bonds. On the basis of this study, we suggest that the prozone phenomenon will occur widely in living cells and that it could be a crucial factor in the regulation of protein complex formation.

The Cellular Level of PR500, a Protein Complex Related to the 19S Regulatory Particle of the Proteasome, Is Regulated in Response to Stresses in Plants

In Arabidopsis seedlings and cauliflower florets, Rpn6 (a proteasome non-ATPase regulatory subunit) was found in two distinct protein complexes of ∼800 and 500 kDa, respectively. The large complex likely represents the proteasome 19S regulator particle (RP) because it displays the expected subunit composition and all characteristics. The small complex, designated PR500, shares at least three subunits with the “lid” subcomplex of 19S RP and is loosely associated with an hsp70 protein. In Arabidopsis COP9 signalosome mutants, PR500 was specifically absent or reduced to an extent that correlates with the severity of the mutations. Furthermore, PR500 was also diminished in response to potential protein-misfolding stresses caused by the heat shock and canavanine treatment. Immunofluorescence studies suggest that PR500 has a distinct localization pattern and is enriched in specific nuclear foci. We propose that PR500 may be evolved in higher plants to cope with the frequently encountered environmental stresses.

Implications of a Developmental-Stage-Dependent Thylakoid-Bound Protease in the Stabilization of the Light-Harvesting Pigment-Protein Complex Serving Photosystem II during Thylakoid Biogenesis in Red Kidney Bean1

Intact etioplasts of bean (Phaseolus vulgaris) plants exhibit proteolytic activity against the exogenously added apoprotein of the light-harvesting pigment-protein complex serving photosystem II (LHCII) that increases as etiolation is prolonged. The activity increases in the membrane fraction but not in the stroma, where it remains low and constant and is mainly directed against LHCII and protochlorophyllide oxidoreductase. The thylakoid proteolytic activity, which is low in etioplasts of 6-d-old etiolated plants, increases in plants pretreated with a pulse of light or exposed to intermittent-light (ImL) cycles, but decreases during prolonged exposure to continuous light, coincident with chlorophyll (Chl) accumulation. To distinguish between the control of Chl and/or development on proteolytic activity, we used plants exposed to ImL cycles of varying dark-phase durations. In ImL plants exposed to an equal number of ImL cycles with short or long dark intervals (i.e. equal Chl accumulation but different developmental stage) proteolytic activity increased with the duration of the dark phase. In plants exposed to ImL for equal durations to such light-dark cycles (i.e. different Chl accumulation but same developmental stage) the proteolytic activity was similar. These results suggest that the protease, which is free to act under limited Chl accumulation, is dependent on the developmental stage of the chloroplast, and give a clue as to why plants in ImL with short dark intervals contain LHCII, whereas those with long dark intervals possess only photosystem-unit cores and lack LHCII.

Structure of the replication terminus-terminator protein complex as probed by affinity cleavage.

The replication terminator protein (RTP) of Bacillus subtilis is a homodimer that binds to each replication terminus and impedes replication fork movement in only one orientation with respect to the replication origin. The three-dimensional structure of the RTP-DNA complex needs to be determined to understand how structurally symmetrical dimers of RTP generate functional asymmetry. The functional unit of each replication terminus of Bacillus subtilis consists of four turns of DNA complexed with two interacting dimers of RTP. Although the crystal structure of the RTP apoprotein dimer has been determined at 2.6-A resolution, the functional unit of the terminus is probably too large and too flexible to lend itself to cocrystallization. We have therefore used an alternative strategy to delineate the three dimensional structure of the RTP-DNA complex by converting the protein into a site-directed chemical nuclease. From the pattern of base-specific cleavage of the terminus DNA by the chemical nuclease, we have mapped the amino acid to base contacts. Using these contacts as distance constraints, with the crystal structure of RTP, we have constructed a model of the DNA-protein complex. The biological implications of the model have been discussed.

Agrobacterium tumefaciens VirB7 and VirB9 form a disulfide-linked protein complex.

Agrobacterium tumefaciens VirB proteins are essential for gene transfer from bacteria to plants. These proteins are postulated to form a transport pore to allow transfer of the T-strand DNA intermediate. To study the function of the VirB proteins in DNA transfer, we developed an expression system in A. tumefaciens. Analysis of one VirB protein, VirB9, by Western blot assays showed that under nonreducing conditions VirB9, when expressed alone, migrates as a approximately 31-kDa band but that it migrates as a approximately 36-kDa band when expressed with all other VirB proteins. The 36-kDa band is converted to the 31-kDa band by the reducing agent 2-mercaptoethanol. Using strains that contain a deletion in a defined virB gene and strains that express specific VirB proteins, we demonstrate that the 36-kDa band is composed of VirB9 and VirB7 that are linked to each other by a disulfide bond. Mutational studies demonstrate that cysteine residues at positions 24 of VirB7 and 262 of VirB9 participate in the formation of this complex.

The SNAP45 subunit of the small nuclear RNA (snRNA) activating protein complex is required for RNA polymerase II and III snRNA gene transcription and interacts with the TATA box binding protein.

The RNA polymerase II and III small nuclear RNA (snRNA) promoters contain a common basal promoter element, the proximal sequence element (PSE). The PSE binds a multisubunit complex we refer to as the snRNA activating protein complex (SNAPc). At least four polypeptides are visible in purified SNAPc preparations, which migrate with apparent molecular masses of 43, 45, 50, and 190 kDa on SDS/polyacrylamide gels. In addition, purified preparations of SNAPc contain variable amounts of TATA box binding protein (TBP). An important question is whether the PSEs of RNA polymerase II and III snRNA promoters recruit the exact same SNAP complex or slightly different versions of SNAPc, differing, for example, by the presence or absence of a subunit. To address this question, we are isolating cDNAs encoding different subunits of SNAPc. We have previously isolated the cDNA encoding the 43-kDa subunit SNAP43. We now report the isolation of the cDNA that encodes the p45 polypeptide. Antibodies directed against p45 retard the mobility of the SNAPc-PSE complex in an electrophoretic mobility shift assay, indicating that p45 is indeed part of SNAPc. We therefore refer to this protein as SNAP45. SNAP45 is exceptionally proline-rich, interacts strongly with TBP, and, like SNAP43, is required for both RNA polymerase II and III transcription of snRNA genes.

A heterotrimeric G protein complex couples the muscarinic m1 receptor to phospholipase C-beta.

We addressed the question as to which subtypes of G protein subunits mediate the activation of phospholipase C-beta by the muscarinic m1 receptor. We used the rat basophilic leukemia cell line RBL-2H3-hm1 stably transfected with the human muscarinic m1 receptor cDNA. We microinjected antisense oligonucleotides into the nuclei of the cells to inhibit selectively the expression of G protein subunits; 48 hr later muscarinic receptors were activated by carbachol, and the increase in free cytosolic calcium concentration ([Ca2+]i) was measured. Antisense oligonucleotides directed against the mRNA coding for alpha(q) and alpha11 subunits both suppressed the carbachol-induced increase in [Ca2+]i. In cells injected with antisense oligonucleotides directed against alpha(o1) and alpha14 subunits, the carbachol effect was unchanged. A corresponding reduction of Galpha(q), and Galpha11 proteins by 70-80% compared to uninjected cells was immunochemically detected 2 days after injection of a mixture of alpha(q) and alpha11 antisense oligonucleotides. Expression of Galpha(q) and Galpha11 completely recovered after 4 days. Cells injected with antisense oligonucleotides directed against the mRNAs encoding for beta1, beta4, and gamma4 subunits showed a suppression of the carbachol-induced increase in [Ca2+]i compared to uninjected cells measured at the same time from the same coverslip, whereas in cells injected with antisense oligonucleotides directed against the beta2, beta3, gamma1, gamma2, gamma3, gamma5, and gamma7 subunits, no suppression of carbachol effect was observed. In summary, the results from RBL-2H3-hm1 cells indicate that the m1 receptor utilizes a G protein complex composed of the subunits alpha(q), alpha11, beta1, beta4, and gamma4 to activate phospholipase C.

An auxiliary factor containing a 240-kDa protein complex is involved in apolipoprotein B RNA editing.

A protein complex involved in apolipoprotein B (apoB) RNA editing, referred to as AUX240 (auxiliary factor containing p240), has been identified through the production of monoclonal antibodies against in vitro assembled 27S editosomes. The 240-kDa protein antigen of AUX240 colocalized with editosome complexes on immunoblots of native gels. Immunoadsorbed extracts were impaired in their ability to assemble editosomes beyond early intermediates and in their ability to edit apoB RNA efficiently. Supplementation of adsorbed extract with AUX240 restored both editosome assembly and editing activities. Several proteins, in addition to p240, ranging in molecular mass from 150 to 45 kDa coimmunopurify as AUX240 under stringent wash conditions. The activity of the catalytic subunit of the editosome APOBEC-1 and mooring sequence RNA binding proteins of 66 and 44 kDa could not be demonstrated in AUX240. The data suggest that p240 and associated proteins constitute an auxiliary factor required for efficient apoB RNA editing. We propose that the role of AUX240 may be regulatory and involve mediation or stabilization of interactions between APOBEC-1 subunits and editing site recognition proteins leading the assembly of the rat liver C/U editosome.

Efficient generation of recombinant adenoviruses using adenovirus DNA-terminal protein complex and a cosmid bearing the full-length virus genome.

An efficient method of constructing recombinant adenoviruses (Ads) has been established. The expression unit to be introduced into recombinant Ad was first inserted into the unique Swa I site of the full-length Ad genome cloned in a cassette cosmid. The cassette bearing the expression unit was then cotransfected into human embryonic kidney 293 cells together with the Ad DNA-terminal protein complex digested at several sites with Eco T22I or Ase I/EcoRI. The use of the parent Ad DNA-terminal protein complex instead of the deproteinized Ad genome DNA allowed very efficient recovery of the desired recombinant Ad, and the above restriction digestion drastically reduced regeneration of the parent virus. Several hundred virus clones were readily obtained in each experiment, and about 70% of the clones were the desired recombinant viruses. Furthermore, because the cassette contained the full-length Ad genome, any position of the genome could be easily modified to develop a new vector design. We established construction systems for two types of Ad vectors, the E1-substitution type and the E4-insertion type. This method may greatly facilitate the application of recombinant Ads and should be useful for further improvement of Ad vectors.

Design and synthesis of novel quinolones directed to the Plasmodium falciparum bc1 protein complex

Tese de Doutoramento, Química, Especialização em Química Orgânica, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2016