Identification of caveolin-1 as a fatty acid binding protein

In an attempt to identify high affinity, fatty acid binding proteins present in 3T3-L1 adipocytes plasma membranes, we labeled proteins in purified plasma membranes with the photoreactive fatty acid analogue, 11-m-diazirinophenoxy[11-3H]undecanoate. A single membrane protein of 22 kDa was covalently labeled after photolysis. This protein fractionated with caveolin-1 containing caveolae and was immunoprecipitated by an anti-caveolin-1 monoclonal antibody. Furthermore, 2D-PAGE analysis revealed that both the alpha and beta isoforms of caveolin-1 could be labeled by the photoreactive fatty acid upon photolysis, indicating that both bind fatty acids. The saturable binding of the photoreactive fatty acid suggests caveolin-1 has a lipid binding site that may either operate during intracellular lipid traffic or regulate caveolin-1 function.     

Sortin2 enhances endocytic trafficking towards the vacuole in Saccharomyces cerevisiae

Background

A highly regulated trafficking of cargo vesicles in eukaryotes performs protein delivery to a variety of cellular compartments of endomembrane system. The two main routes, the secretory and the endocytic pathways have pivotal functions in uni- and multi-cellular organisms. Protein delivery and targeting includes cargo recognition, vesicle formation and fusion. Developing new tools to modulate protein trafficking allows better understanding the endomembrane system mechanisms and their regulation. The compound Sortin2 has been described as a protein trafficking modulator affecting targeting of the vacuolar protein carboxypeptidase Y (CPY), triggering its secretion in Saccharomyces cerevisiae.

Results

A reverse chemical-genetics approach was used to identify key proteins for Sortin2 bioactivity. A genome-wide Sortin2 resistance screen revealed six yeast deletion mutants that do not secrete CPY when grown at Sortin2 condition where the parental strain does: met18, sla1, clc1, dfg10, dpl1 and yjl175w. Integrating mutant phenotype and gene ontology annotation of the corresponding genes and their interactome pointed towards a high representation of genes involved in the endocytic process. In wild type yeast endocytosis towards the vacuole was faster in presence of Sortin2, which further validates the data of the genome-wide screen. This effect of Sortin2 depends on structural features of the molecule, suggesting compound specificity. Sortin2 did not affect endocytic trafficking in Sortin2-resistant mutants, strongly suggesting that the Sortin2 effects on the secretory and endocytic pathways are linked.

Conclusions

Overall, the results reveal that Sortin2 enhances the endocytic transport pathway in Saccharomyces cerevisiae. This cellular effect is most likely at the level where secretory and endocytic pathways are merged. Them Sortin2 specificity over the endomembrane system places it as a powerful biological modulator for cell biology.

Electronic supplementary material

The online version of this article (doi:10.1186/s40659-015-0032-9) contains supplementary material, which is available to authorized users.     

Study of interaction of XRCC1 with DNA and proteins of base excision repair by photoaffinity labeling technique

The X-ray repair cross-complementing group 1 (XRCC1) protein plays a central role in base excision repair (BER) interacting with and modulating activity of key BER proteins. To estimate the influence of XRCC1 on interactions of BER proteins poly(ADP-ribose) polymerase 1 (PARP1), apurinic/apyrimidinic endonuclease 1 (APE1), flap endonuclease 1 (FEN1), and DNA polymerase beta (Pol beta) with DNA intermediates, photoaffinity labeling using different photoreactive DNA was carried out in the presence or absence of XRCC1. XRCC1 competes with APE1, FEN1, and PARP1 for DNA binding, while Pol beta increases the efficiency of XRCC1 modification. To study the interactions of XRCC1 with DNA and proteins at the initial stages of BER, DNA duplexes containing a photoreactive group in the template strand opposite the damage were designed. DNA duplexes with 8-oxoguanine or dihydrothymine opposite the photoreactive group were recognized and cleaved by specific DNA glycosylases (OGG1 or NTH1, correspondingly), although the rate of oxidized base excision in the photoreactive structures was lower than in normal substrates. XRCC1 does not display any specificity in recognition of DNA duplexes with damaged bases compared to regular DNA. A photoreactive group opposite a synthetic apurinic/apyrimidinic (AP) site (3-hydroxy-2-hydroxymethyltetrahydrofuran) weakly influences the incision efficiency of AP site analog by APE1. In the absence of magnesium ions, i.e. when incision of AP sites cannot occur, APE1 and XRCC1 compete for DNA binding when present together. However, in the presence of magnesium ions the level of XRCC1 modification increased upon APE1 addition, since APE1 creates nicked DNA duplex, which interacts with XRCC1 more efficiently.     

Binary system for selective photoaffinity labeling of base excision repair DNA polymerases

A system of photoaffinity reagents for selective labeling of DNA polymerases in extracts has been examined. To create the photoreactive DNA probe in situ, DNA substrates containing a synthetic abasic site are incubated in mouse embryonic fibroblast (MEF) cellular extract in the presence of base-substituted arylazido derivatives of dNTPs. This results in synthesis of a photoreactive long patch base excision repair (BER) intermediate. The arylazido photoreactive group is then activated through energy transfer from the pyrene group of a dNTP analog (Pyr-dUTP), following 365 nm UV light exposure. Pyr-dUTP binds to the active site of DNA polymerases, and the pyrene group, when excited by 365 nm UV light, activates the nearby photoreactive group in the BER intermediate resulting in crosslinking of DNA-bound DNA polymerases. Under these conditions, various DNA binding proteins that are unable to bind Pyr-dUTP are not crosslinked to DNA. DNA polymerase β is the predominant crosslinked protein observed in the MEF extract. In contrast, several other DNA binding proteins are labeled under conditions of direct UV light activation of the photoreactive group at 312 nm. This study illustrates use of a new method of selective labeling of DNA polymerases in a crude cellular extract.     

Iodinated photoreactive vasopressin antagonists: labelling of hepatic vasopressin receptor subunits

To identify and characterize V1 vasopressin receptors, photoreactive antagonists of the glycogenolytic and vasoconstrictor activity of vasopressin have been synthesized. The following analogues with 3-mercapto-3,3-cyclopentamethylene-propionic acid (Mca) and N-methylalanine (MeAla) in position 1 and 7 of vasopressin (VP) were effective V1 antagonists: [Mca1, D-Tyr2, MeAla7, Lys8]VP (1), [Mca1, MeAla7, Arg8, Lys9]VP (2), [Mca1, MeAla7, Arg8, D-Lys9]VP (3). Introduction of the photoreactive 4-azidophenylamidino group into the side-chain of Lys8 in analogue 1 or into Lys9 in analogues 2 and 3 increased the potency (for analogue 1 a tenfold increase in the antiglycogenolytic effect and a fivefold increase in the antivasopressor effect) and binding affinity for the rat hepatic V1 receptor. Mono-iodination at Tyr2 with 125I resulted in photoreactive antagonists of high specific radioactivity, which had roughly the same binding affinity as vasopressin for the rat hepatic V1 receptor (Kd = 0.9-1.8 nM). In photoaffinity labelling experiments with purified rat liver membranes, containing 2--3 pmol V1 receptor/mg protein, the analogues labelled specifically two proteins with the relative molecular masses (Mr) of 30,000 and 38,000. These results and the results of a recent study using 3H-labelled photoreactive vasopressin agonists [Boer, R. and Fahrenholz, F. (1985) J. Biol. Chem. 260, 15051-15054] provide evidence that both vasopressin agonists and antagonists can interact with the same two subunits of the heterodimeric hepatic V1 receptor. Furthermore the radioiodinated photoreactive V1 antagonists should be helpful to identify V1 receptor proteins in membranes of other cell types.     

Affinity labeling of flap-endonuclease FEN-1 by photoreactive DNAs

Eukaryotic flap-endonuclease (FEN-1) is 42-kD single-subunit structure-specific nuclease that cleaves 5"-flap strands of the branched DNA structure and possesses 5"-exonuclease activity. FEN-1 participates in DNA replication, repair, and recombination. The interaction of FEN-1 with DNA structures generated during replication and repair was studied using two types of photoreactive oligonucleotides. Oligonucleotides bearing a photoreactive arylazido group at the 3"-end of the primer were synthesized in situ by the action of DNA polymerase using base-substituted photoreactive dUTP analogs as the substrates. The photoreactive group was also bound to the 5"-end phosphate group of the oligonucleotide by chemical synthesis. Interaction of FEN-1 with both 5"- and 3"-ends of the nick or with primer–template systems containing 5"- or 3"-protruding DNA strands was shown. Formation of a structure with the 5"-flap containing the photoreactive group results in decrease of the level of protein labeling caused by cleavage of the photoreactive group due to FEN-1 endonuclease activity. Photoaffinity labeling of proteins of mouse fibroblast cell extract was performed using the radioactively labeled DNA duplex with the photoreactive group at the 3"-end and the apurine/apyrimidine site at the 5"-end of the nick. This structure is a photoreactive analog of an intermediate formed during DNA repair and was generated by the action of cell enzymes from the initial DNA duplex containing the 3-hydroxy-2-hydroxymethyltetrahydrofurane residue. FEN-1 is shown to be one of the photolabeled proteins; this indicates possible participation of this enzyme in base excision repair.     

A new highly efficient photoreactive analogue of dCTP. Synthesis, characterization, and application in photoaffinity modification of DNA binding proteins

A new base-substituted analogue of dCTP, exo-N-{2-[N-(4-azido-2,5-difluoro-3-chloropyridine-6-yl)-3-aminopropionyl]aminoethyl}-2'-deoxycytidine-5'-triphosphate (FAP-dCTP) has been synthesized and characterized. FAP-dCTP is an efficient substrate of mammalian DNA polymerase beta in the reaction of primer elongation displaying substrate properties as an analogue of dCTP and dTTP. FAP-dCTP was used for the photoaffinity modification of mammalian DNA polymerase beta. Two approaches to photoaffinity labeling were utilized. In one approach, photoreactive FAP-dCTP was first incorporated into radiolabeled primer-template, and photoreactive DNA was UV-irradiated in the presence of DNA polymerase beta, which resulted in the polymerase labeling by photoreactive primer. In an alternate approach, FAP-dCTP was first UV-cross-linked to the enzyme; subsequently, radiolabeled primer-template was added, and the enzyme-linked FAP-dCTP was incorporated into the 3'-end of radioactive primer. This "catalytic" modification pathway was shown to be less specific in recognition of FAP-dCTP as an analogue of dCTP than dTTP. FAP-dCTP was used as substrate of endogenous DNA polymerases of HeLa cell extract to synthesize photoreactive DNAs for photoaffinity modification of cell proteins. UV irradiation results in modification of DNA binding proteins of cell extract. The level of photoaffinity labeling of protein targets in the cell extract was strongly dependent on the efficiency of synthesis of photoreactive DNA.     

The effect of structural differences in the reducing terminus of sugars on the binding affinity of carbohydrates and proteins analyzed using photoaffinity labeling

Because carbohydrates and proteins bind with such low affinity, the nature of their interactions is not clear. Photoaffinity labeling with diazirin groups is useful for elucidating the roles of carbohydrates in these binding processes. However, when carbohydrate probes are synthesized according to this conventional method, the reducing terminus of the sugar is opened to provide an acyclic structure. Because greater elucidation of carbohydrate-protein interactions requires a closed-ring carbohydrate in addition to the photoreactive group, we synthesized new molecular tools. The carbohydrate ligands were synthesized in three steps (glycosylation with allyl alcohol, deprotection, and ozonolysis). Specific binding proteins for carbohydrate ligands were obtained by photoaffinity labeling. Closed ring-type carbohydrate ligands, in which the reducing sugar is closed, bound to lectins more strongly than open ring-type sugars. Carbohydrate to protein binding was observed using AFM.     

Photoaffinity labeling and fatty acid permeation in 3T3-L1 adipocytes

Long chain fatty acid uptake was investigated in 3T3-L1 cells. Differentiation of these cells from fibroblasts to adipocytes was accompanied by an 8.5-fold increase in the rate of oleate uptake. This was saturable in adipocytes with apparent Kt and Vmax values of 78 nM and 16 nmol/min/mg cell protein, respectively. A number of proteins in various subcellular fractions of differentiated cells were labeled with the photoreactive fatty acid 11-m-diazirinophenoxy[11-3H]undecanoate. A 15-kDa cytoplasmic protein was induced upon differentiation to adipocytes. This protein was labeled with the photoreactive fatty acid in cytoplasm isolated from differentiated adipocytes, but not in cytoplasm from undifferentiated, fibroblastic cells. Furthermore, a high affinity fatty acid binding protein of 22 kDa was identified in plasma membranes of undifferentiated cells, and its level of labeling increased 2-fold upon differentiation. These results indicate the usefulness of the photoreactive fatty acid in identifying cellular fatty acid binding proteins, and its potential to elucidate the spatial and temporal distribution of fatty acids in intact cells.     

The ND1 subunit constructs the inhibitor binding domain in bovine heart mitochondrial complex I

The inhibitor binding domain in bovine complex I is believed to be constructed by multisubunits, but it remains to be learned how the binding positions of chemically diverse inhibitors relate to each other. To get insight into the inhibitor binding domain in complex I, we synthesized a photoreactive acetogenin [[125I](trifluoromethyl)phenyldiazirinylacetogenin, [125I]TDA], in which an aryldiazirine group serves as both a photoreactive group and a substitute for the gamma-lactone ring that is a common toxophore of numerous natural acetogenins, and carried out photoaffinity labeling to identify the labeled subunit using bovine heart submitochondrial particles (SMP). When SMP were UV-irradiated in the presence of [125I]TDA, radioactivity was predominantly incorporated into an approximately 30 kDa band on a SDS gel. Blue native gel electrophoresis of the [125I]TDA-labeled SMP revealed that the majority of radioactivity was observed in complex I. Analysis of complex I on a SDS gel showed a predominant peak of radioactivity at approximately 30 kDa. Immnoprecipitation of the [125I]TDA-labeled complex I with anti-bovine ND1 antibody indicated that the labeled protein is the ND1 subunit. A variety of complex I inhibitors such as piericidin A and rotenone efficiently suppressed the specific binding of [125I]TDA to ND1, indicating that they share a common binding domain. However, the suppression efficiency of Deltalac-acetogenin, a new type of complex I inhibitor synthesized in our laboratory, was much lower than that of the traditional inhibitors. Our results unequivocally reveal that the ND1 subunit constructs the inhibitor binding domain, though the contribution of this subunit has been challenged. Further, the present study corroborates our previous proposition that the inhibition site of Deltalac-acetogenins differs from that of traditional inhibitors.     

Differential labeling of the alpha and beta 1 subunits of the sodium channel by photoreactive derivatives of scorpion toxin

The separation of two photoreactive derivatives of the alpha-scorpion toxin from Leiurus quinquestriatus is described. When the two photoreactive derivatives were photolyzed separately in the presence of brain membranes containing voltage-sensitive sodium channels, one labeled the alpha subunit preferentially while the other labeled beta 1 more intensely than alpha. Batrachotoxin enhanced the efficiency of covalent labeling by the photoreactive derivatives of scorpion toxin. In all the labeling experiments, the specific incorporation of radioactive scorpion toxin was eliminated by an excess of nonradioactive scorpion toxin. The alpha polypeptide labeled in synaptosomes by photoreactive scorpion toxin was demonstrated by immunological techniques to be the same large polypeptide identified in sodium channels purified by their saxitoxin binding activity. The alpha and beta 1 subunits were detected by rapid photoaffinity labeling of a freshly prepared brain homogenate in the presence of a mixture of nine protease inhibitors, indicating that they are components of the sodium channel in intact brain tissue. The association of the covalently labeled polypeptides with the membrane was investigated by treatment of labeled synaptosomes with various agents known to remove proteins only indirectly attached to the lipid bilayer via a membrane-bound protein. In all cases, both the alpha and the beta 1 polypeptides remained in the membrane fraction following extraction. This confirms earlier proposals that the alpha polypeptide has a portion of its mass embedded within the lipid bilayer and suggests that the beta 1 polypeptide does as well.     

Photoaffinity labeling of proteins in bovine testis nuclear extract

A binary system of photoaffinity reagents for selective affinity labeling of DNA polymerases has been developed. The photoreactive probe was formed in nuclear extract, using an end-labeled oligonucleotide containing a synthetic abasic site. This site was incised by apurinic/apyrimidinic endonuclease and then dNMPs carrying a photoreactive adduct were added to the 3(') hydroxyl using base-substituted arylazido derivatives of dUTP or dCTP. This results in the synthesis of photoreactive base excision repair (BER) intermediates. The photoreactive group was then activated, either directly (UV light exposure 320nm) or in the presence of the sensitizer of dTTP analog containing a pyrene group (Pyr-dUTP) under UV light 365nm. DNA polymerase beta was the main target crosslinked by photoreactive BER intermediates in this nuclear extract. In contrast, several proteins were labeled under the conditions of direct activation of arylazido group.     

Strand-specific binding of RPA and XPA to damaged duplex DNA

The nucleotide excision repair (NER) pathway is a major pathway used to repair bulky adduct DNA damage. Two proteins, xeroderma pigmentosum group A protein (XPA) and replication protein A (RPA), have been implicated in the role of DNA damage recognition in the NER pathway. The particular manner in which these two damage recognition proteins align themselves with respect to a damaged DNA site was assessed using photoreactive base analogues within specific DNA substrates to allow site-specific cross-linking of the damage recognition proteins. Results of these studies demonstrate that both RPA and XPA are in close proximity to the adduct as measured by cross-linking of each protein directly to the platinum moiety. Additional studies demonstrate that XPA contacts both the damaged and undamaged strands of the duplex DNA. Direct evidence is presented demonstrating preferential binding of RPA to the undamaged strand of a duplex damaged DNA molecule.     

Photoaffinity labeling of fatty acid-binding proteins involved in long chain fatty acid transport in Escherichia coli.

The photoreactive fatty acid 11-m-diazirinophenoxy-[11-3H]undecanoate was shown to be taken up specifically by the fatty acid transport system expressed in Escherichia coli grown on oleate. This photoreactive fatty acid analogue was therefore used to identify proteins involved in fatty acid uptake in E. coli. The fadL protein was labeled by the probe, confirmed to be exclusively in the outer membrane and to exhibit the heat modifiable behavior typical of outer membrane proteins. The apparent pI of the incompletely denatured form of the protein having the mobility of a 33-kDa protein was 4.6 while that of the fully denatured form was consistent with the calculated value of 5.2. The denaturation was reversible depending upon the protein to detergent ratios. The photoreactive fatty acid partitions into the outer membrane, resulting in extensive photolabeling of the lipid; a high affinity fatty acid-binding site is not apparent in total membranes labeled using free fatty acids due to this large binding capacity of the outer membrane. However, when the free fatty acid concentration was controlled by supplying it as a bovine serum albumin complex, the fadL protein exhibited saturable high affinity fatty acid binding, having an apparent Kd for the probe of 63 nM. The methods described very readily identify fatty acid-binding proteins: the fact that even when the sensitivity was increased 500-fold, no evidence was found for the presence of a fatty acid-binding protein in the inner membrane is consistent with the proposal that fatty acid permeation across the plasma membrane is not protein mediated but occurs by a simple diffusive mechanism.     

Photoaffinity labelling of the oxytocin receptor in plasma membranes from rat mammary gland

Plasma membranes from rat mammary gland containing a high concentration of [3H]oxytocin binding sites (2.8 pmol/mg protein) were used for photoaffinity labelling experiments. Competitive binding experiments show that these receptors bind with high affinity the specific oxytocin agonist [Thr4, Sar7]oxytocin and the analogue of 1-deamino-[8-lysine]vasopressin containing a photoreactive azidobenzoyl group (Abz) at the side chain of lysine. The tritium-labelled (50 Ci/mol) photoreactive analogue incorporated into a membrane protein with an apparent relative molecular mass of 65,000 +/- 3000 Da (n = 16). The labelling of this protein was completely suppressed by an excess of oxytocin.     

Synthesis and characterization of a radioiodinated, photoreactive and physiologically active analogue of platelet activating factor

The multistep synthesis of a photoreactive, radioactive and aggregating analogue of platelet-activating factor (PAF)-acether is described. The photoreactive and radioactive moiety was added at the last step; the specific radioactivity was higher than 1000 Ci/mmol. The concentration of this new analogue which causes 50% of aggregation of platelets were of the same order of magnitude as for synthetic snPAF-acether, so as for two other analogues having a bulky group at the omega end of the fatty ether chain. The photoreactivity was proved by the covalent binding of the analogue to protein (BSA) after 10-min irradiation times at 300 nm. The binding was largely prevented by prior (not by later) addition of a high concentration of lyso phosphatidyl choline.     

Structure of photoreactive binary system of oligonucleotide conjugates assembled on the target nucleotide sequence

Recently we have developed an approach to superspecific photomodification of nucleic acids by binary systems of oligonucleotides conjugated to precursor groups capable of assembling into photoactivatable structure upon simultaneous binding of the conjugates to the target. We have investigated the solution structure of a model binary system 1:2:3, where 1 is the target 12-mer 5'-pdGTATCAGTTTCT, 2 is the photoreactive conjugate 5'-dAGAAACp-NH(CH2)2NH-Az and 3 is the sensitizing conjugate 5'-Pyr-pdTGATAC (Az is p-azidotetrafluorobenzoyl group and Pyr is the pyrenyl-1-methylamino group). The photoreaction within this complex results in crosslinking of reagent 2 with N7-position of the G7 residue of the target thus indicating that the photoreactive Az residue is located in the major groove near the G7 residue. The center-to-center distances between the Pyr and Az moieties in complex 1:2:3 independently determined by the Pyr-group fluorescence quenching and the Az-group sensitized photodecomposition were 11.2 and 12.6 A, respectively.     

Interaction of nucleotide excision repair factors XPC-HR23B, XPA, and RPA with damaged DNA

The interaction of nucleotide excision repair factors--xeroderma pigmentosum complementation group C protein in complex with human homolog of yeast Rad23 protein (XPC-HR23B), replication protein A (RPA), and xeroderma pigmentosum complementation group A protein (XPA)--with 48-mer DNA duplexes imitating damaged DNA structures was investigated. All studied proteins demonstrated low specificity in binding to damaged DNA compared with undamaged DNA duplexes. RPA stimulates formation of XPC-HR23B complex with DNA, and when XPA and XPC-HR23B are simultaneously present in the reaction mixture a synergistic effect in binding of these proteins to DNA is observed. RPA crosslinks to DNA bearing photoreactive 5I-dUMP residue on one strand and fluorescein-substituted dUMP analog as a lesion in the opposite strand of DNA duplex and also stimulates cross-linking with XPC-HR23B. Therefore, RPA might be one of the main regulation factors at various stages of nucleotide excision repair. The data are in agreement with the cooperative binding model of nucleotide excision repair factors participating in pre-incision complex formation with DNA duplexes bearing damages.     

Interaction of Replication Protein A with Photoreactive DNA Structures

A new photoreactive oligonucleotide derivative was synthesized with a perfluoroarylazido group attached to the 2'-position of the ribose fragment of the 5'-terminal nucleotide. Using this conjugate, photoreactive DNA duplexes were produced which contained single-stranded regions of different length, single-stranded breaks (nicks), and also ds duplex with a photoreactive group inside one of the chains. These structures imitate DNA intermediates generated at different stages of DNA replication and repair. The interaction of replication protein A (RPA) with the resulting DNA structures was studied using photoaffinity modification and gel retardation assay. Independently of the DNA structure, only the large subunit of RPA (p70) was crosslinked to photoreactive DNAs, and the intensity of its labeling increased with decrease in the size of the single-stranded region and was maximal in the case of the nick-containing DNA structure. By gel retardation, the most effective binding of RPA to this structure was shown, whereas the complexing of RPA with DNA containing the unmodified nick and also with the full duplex containing the photoreactive group inside the chain was significantly less effective. The data suggest that RPA should be sensitive to such damages in the double-stranded DNA structure.     

Design and synthesis of small molecule-conjugated photoaffinity nanoprobes for a streamlined analysis of binding proteins

We designed and synthesized a set of photoaffinity nanoprobes, which multivalently display a small molecule ligand and a photoreactive group on gold nanoparticles. Due to the typically hydrophobic nature of these two functional groups, a hydrophilic spacer was additionally introduced to co-functionalize the nanoprobes to maintain their dispersibility in aqueous buffer solutions. Photoaffinity labeling studies using the nanoprobes composed of different ratios of three functional groups showed that including high density of the spacer group attenuates crosslinking efficiency. Comparative analysis of the reactivity among three major photoreactive groups suggested that unlike in the context of conventional photoaffinity probes, arylazide group enables the most selective crosslinking of a model small molecule binding protein.