Characterization of low affinity Fcγ receptor biotinylation under controlled reaction conditions by mass spectrometry and ligand binding analysis

Chemical protein biotinylation and streptavidin or anti‐biotin‐based capture is regularly used for proteins as a more controlled alternative to direct coupling of the protein on a biosensor surface. On biotinylation an interaction site of interest may be blocked by the biotin groups, diminishing apparent activity of the protein. Minimal biotinylation can circumvent the loss of apparent activity, but still a binding site of interest can be blocked when labeling an amino acid involved in the binding. Here, we describe reaction condition optimization studies for minimal labeling. We have chosen low affinity Fcγ receptors as model compounds as these proteins contain many lysines in their active binding site and as such provide an interesting system for a minimal labeling approach. We were able to identify the most critical parameters (protein:biotin ratio and incubation pH) for a minimal labeling approach in which the proteins of choice remain most active toward analyte binding. Localization of biotinylation by mass spectrometric peptide mapping on minimally labeled material was correlated to protein activity in binding assays. We show that only aiming at minimal labeling is not sufficient to maintain an active protein. Careful fine‐tuning of critical parameters is important to reduce biotinylation in a protein binding site.     

Recognition of a bicoid mRNA localization signal by a protein complex containing Swallow,Nod, and RNA binding proteins

Localization of mRNAs, a process essential for embryonic body patterning in Drosophila, requires recognition of cis-acting signals by cellular components responsible for movement and anchoring. We have purified a large multiprotein complex that binds a minimal form of the bicoid mRNA localization signal in a manner both specific and sensitive to inactivating mutations. Identified complex components include the RNA binding proteins Modulo, PABP, and Smooth, the known localization factor Swallow, and the kinesin family member Nod. We demonstrate that localization of bcd mRNA is defective in modulo mutants. The presence of three required localization components (Swallow, Modulo, and specific RNA binding activity) within the recognition complex strongly implicates it in mRNA localization.     

ATP-regulated interactions between P1 ParA, ParB and non-specific DNA that are stabilized by the plasmid partition site, parS

Localization of the P1 plasmid requires two proteins, ParA and ParB, which act on the plasmid partition site, parS. ParB is a site-specific DNA-binding protein and ParA is a Walker-type ATPase with non-specific DNA-binding activity. In vivo ParA binds the bacterial nucleoid and forms dynamic patterns that are governed by the ParB-parS partition complex on the plasmid. How these interactions drive plasmid movement and localization is not well understood. Here we have identified a large protein-DNA complex in vitro that requires ParA, ParB and ATP, and have characterized its assembly by sucrose gradient sedimentation and light scattering assays. ATP binding and hydrolysis mediated the assembly and disassembly of this complex, while ADP antagonized complex formation. The complex was not dependent on, but was stabilized by, parS. The properties indicate that ParA and ParB are binding and bridging multiple DNA molecules to create a large meshwork of protein-DNA molecules that involves both specific and non-specific DNA. We propose that this complex represents a dynamic adaptor complex between the plasmid and nucleoid, and further, that this interaction drives the redistribution of partition proteins and the plasmid over the nucleoid during partition.     

Localization of FtsI (PBP3) to the Septal Ring Requires Its Membrane Anchor, the Z Ring, FtsA, FtsQ, and FtsL

Assembly of the division septum in bacteria is mediated by several proteins that localize to the division site. One of these, FtsI (also called penicillin-binding protein 3) of Escherichia coli, consists of a short cytoplasmic domain, a single membrane-spanning segment, and a large periplasmic domain that encodes a transpeptidase activity involved in synthesis of septal peptidoglycan. We have constructed a merodiploid strain with a wild-type copy of ftsI at the normal chromosomal locus and a genetic fusion of ftsI to the green fluorescent protein (gfp) at the lambda attachment site. gfp-ftsI was expressed at physiologically appropriate levels under control of a regulatable promoter. Consistent with previous results based on immunofluorescence microscopy GFP-FtsI localized to the division site during the later stages of cell growth and throughout septation. Localization of GFP-FtsI to the cell pole(s) was not observed unless the protein was overproduced about 10-fold. Membrane anchor alterations shown previously to impair division but not membrane insertion or transpeptidase activity were found to interfere with localization of GFP-FtsI to the division site. In contrast, GFP-FtsI localized well in the presence of β-lactam antibiotics that inhibit the transpeptidase activity of FtsI. Septal localization depended upon every other division protein tested (FtsZ, FtsA, FtsQ, and FtsL). We conclude that FtsI is a late recruit to the division site, and that its localization depends on an intact membrane anchor.     

Coordination of endoplasmic reticulum and mRNA localization to the yeast bud

Localization of messenger RNAs and local protein synthesis contribute to asymmetric protein distribution not only of cytoplasmic but also of membrane or secreted proteins. Since synthesis of the latter protein classes occurs at the rough endoplasmic reticulum (ER), mRNA localization and distribution of ER should be coordinated. However, this coordination is not yet understood. In yeast, mRNA localization to the growing bud depends on the myosin Myo4p, its adaptor She3p, and the specific RNA binding protein She2p. These proteins mediate the localization of 23 mRNAs including ASH1 mRNA and mRNAs encoding membrane proteins. In addition, Myo4p and She3p are required for segregation of cortical ER to the bud. Here we show, with ASH1 mRNA as a model mRNA, that localizing messenger ribonucleoprotein (mRNP) particles comigrate with tubular ER structures to the bud, which requires the RNA binding protein She2p. Coordinated movement of the ASH1 mRNP with ER tubules but not their association with each other depends on Myo4p and She3p. Subcellular fractionation experiments demonstrate a cosegregation of ER and She2p, which is independent of Myo4p, She3p, or polysomes. Our findings suggest a novel model for mRNA localization that involves association of She2p and mRNPs with ER tubules and myosin-dependent cotransport of tubules and localized mRNPs.     

Characterization of GTP binding and hydrolysis in plasma membranes of zucchini.

We have investigated the possibility that G-protein-like entities may be present in the plasma membrane (PM) of zucchini (Cucurbita pepo L.) hypocotyls by examining a number of criteria common to animal and yeast G-proteins. The GTP binding and hydrolysis characteristics of purified zucchini PM are similar to the characteristics of a number of known G-proteins. Our results demonstrate GTP binding to a single PM site having a Kd value between 16-31 nM. This binding has a high specificity for guanine nucleotides, and is stimulated by Mg2+, detergents, and fluoride or aluminium ions. The GTPase activity (Km = 0.49 micromole) of zucchini PM shows a sensitivity to NaF similar to that seen for other G-proteins. Localization of GTP mu 35S binding to nitrocellulose blots of proteins separated by SDS-PAGE indicates a 30-kDa protein as the predominant GTP-binding species in zucchini PM. Taken together, these data indicate that plant PM contains proteins which are biochemically similar to previously characterized G-proteins.     

C-terminal WxL domain mediates cell wall binding in Enterococcus faecalis and other gram-positive bacteria

Analysis of the genome sequence of Enterococcus faecalis clinical isolate V583 revealed novel genes encoding surface proteins. Twenty-seven of these proteins, annotated as having unknown functions, possess a putative N-terminal signal peptide and a conserved C-terminal region characterized by a novel conserved domain designated WxL. Proteins having similar characteristics were also detected in other low-G+C-content gram-positive bacteria. We hypothesized that the WxL region might be a determinant of bacterial cell location. This hypothesis was tested by generating protein fusions between the C-terminal regions of two WxL proteins in E. faecalis and a nuclease reporter protein. We demonstrated that the C-terminal regions of both proteins conferred a cell surface localization to the reporter fusions in E. faecalis. This localization was eliminated by introducing specific deletions into the domains. Interestingly, exogenously added protein fusions displayed binding to whole cells of various gram-positive bacteria. We also showed that the peptidoglycan was a binding ligand for WxL domain attachment to the cell surface and that neither proteins nor carbohydrates were necessary for binding. Based on our findings, we propose that the WxL region is a novel cell wall binding domain in E. faecalis and other gram-positive bacteria.     

Imp associates with squid and Hrp48 and contributes to localized expression of gurken in the oocyte

Localization and translational control of Drosophila melanogaster gurken and oskar mRNAs rely on the hnRNP proteins Squid and Hrp48, which are complexed with one another in the ovary. Imp, the Drosophila homolog of proteins acting in localization of mRNAs in other species, is also associated with Squid and Hrp48. Notably, Imp is concentrated at sites of gurken and oskar mRNA localization in the oocyte, and alteration of gurken localization also alters Imp distribution. Imp binds gurken mRNA with high affinity in vitro; thus, the colocalization with gurken mRNA in vivo is likely to be the result of direct binding. Imp mutants support apparently normal regulation of gurken and oskar mRNAs. However, loss of Imp activity partially suppresses a gurken misexpression phenotype, indicating that Imp does act in control of gurken expression but has a largely redundant role that is only revealed when normal gurken expression is perturbed. Overexpression of Imp disrupts localization of gurken mRNA as well as localization and translational regulation of oskar mRNA. The opposing effects of reduced and elevated Imp activity on gurken mRNA expression indicate a role in gurken mRNA regulation.     

HIV-1 Replication and APOBEC3 Antiviral Activity Are Not Regulated by P Bodies

The APOBEC3 cytidine deaminases play a critical role in host-mediated defense against exogenous viruses, most notably, human immunodeficiency virus type-1 (HIV-1) and endogenous transposable elements. APOBEC3G and APOBEC3F interact with numerous proteins that regulate cellular RNA metabolism, including components of the RNA-induced silencing complex (RISC), and colocalize with a subset of these proteins to mRNA processing bodies (P bodies), which are sites of mRNA translational repression and decay. We sought to determine the role of P bodies and associated proteins in HIV-1 replication and APOBEC3 antiviral activity. While we established a positive correlation between APOBEC3 protein incorporation into virions and localization to P bodies, depletion of the P-body components DDX6 or Lsm1 did not affect HIV-1 replication, APOBEC3 packaging into virions or APOBEC3 protein mediated inhibition of HIV-1 infectivity. In addition, neither HIV-1 genomic RNA nor Gag colocalized with P-body proteins. However, simultaneous depletion of multiple Argonaute family members, the effector proteins of RISC, could modestly increase viral infectivity. Because some APOBEC3 proteins interact with several Argonaute proteins, we also tested whether they could modulate microRNA (miRNA) activity. We found no evidence for the specific regulation of miRNA function by the APOBEC3 proteins, though more general effects on transfected gene expression were observed. In sum, our results indicate that P bodies and certain associated proteins do not regulate HIV-1 replication or APOBEC3 protein antiviral activity. Localization to P bodies may therefore provide a means of sequestering APOBEC3 enzymatic activity away from cellular DNA or may be linked to as yet unidentified cellular functions.     

Localization of organelle proteins by isotope tagging (LOPIT)

We describe a proteomics method for determining the subcellular localization of membrane proteins. Organelles are partially separated using centrifugation through self-generating density gradients. Proteins from each organelle co-fractionate and therefore exhibit similar distributions in the gradient. Protein distributions can be determined through a series of pair-wise comparisons of gradient fractions, using cleavable ICAT to enable relative quantitation of protein levels by MS. The localization of novel proteins is determined using multivariate data analysis techniques to match their distributions to those of proteins that are known to reside in specific organelles. Using this approach, we have simultaneously demonstrated the localization of membrane proteins in both the endoplasmic reticulum and the Golgi apparatus in Arabidopsis. Localization of organelle proteins by isotope tagging is a new tool for high-throughput protein localization, which is applicable to a wide range of research areas such as the study of organelle function and protein trafficking.     

Eukaryotic localization, activation and ubiquitinylation of a bacterial type III secreted toxin

Type III secretion is a widespread method whereby Gram-negative bacteria introduce toxins into eukaryotic cells. These toxins mimic or subvert a normal cellular process by interacting with a specific target, although how toxins reach their site of action is unclear. We set out to investigate the intracellular localization of a type III toxin of Pseudomonas aeruginosa called ExoU, which has phospholipase activity and requires a eukaryotic factor for activity. We found that ExoU is localized to the plasma membrane and undergoes modification within the cell by addition of two ubiquitin molecules at lysine-178. A region of five amino acids at position 679-683 near the C-terminus of the ExoU protein controls both membrane localization and ubiquitinylation. Site-directed mutagenesis identified a tryptophan at position 681 as crucial for these effects. We found that the same region at position 679-683 was also required for cell toxicity produced by ExoU as well as in vitro phospholipase activity. Localization of the phospholipase ExoU to the plasma membrane is thus required for activation and allows efficient utilization of adjacent substrate phospholipids.     

Regulation of yeast Yak1 kinase by PKA and autophosphorylation-dependent 14-3-3 binding

Yak1 is a member of an evolutionarily conserved family of Ser/Thr protein kinases known as dual-specificity Tyr phosphorylation-regulated kinases (DYRKs). Yak1 was originally identified as a growth antagonist, which functions downstream of Ras/PKA signalling pathway. It has been known that Yak1 is phosphorylated by PKA in vitro and is translocated to the nucleus upon nutrient deprivation. However, the regulatory mechanisms for Yak1 activity and localization are largely unknown. In the present study, we investigated the role of PKA and Bmh1, a yeast 14-3-3 protein, in regulation of Yak1. We demonstrate that PKA-dependent phosphorylation of Yak1 on Ser295 and two minor sites inhibits nuclear localization of Yak1. We also show that intramolecular autophosphorylation on at least four Ser/Thr residues in the non-catalytic N-terminal domain is required for full kinase activity of Yak1. The most potent autophosphorylation site, Thr335, plays an essential role for Bmh1 binding in collaboration with a yet unidentified second binding site in the N-terminal domain. Bmh1 binding decreases the catalytic activity of Yak1 without affecting its subcellular localization. Since the binding of 14-3-3 proteins to Yak1 coincides with PKA activity, such regulatory mechanisms might allow cytoplasmic retention of an inactive form of Yak1 under high glucose conditions.     

Primary structure and binding activity of the hnRNP U protein: binding RNA through RGG box.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are thought to influence the structure of hnRNA and participate in the processing of hnRNA to mRNA. The hnRNP U protein is an abundant nucleoplasmic phosphoprotein that is the largest of the major hnRNP proteins (120 kDa by SDS-PAGE). HnRNP U binds pre-mRNA in vivo and binds both RNA and ssDNA in vitro. Here we describe the cloning and sequencing of a cDNA encoding the hnRNP U protein, the determination of its amino acid sequence and the delineation of a region in this protein that confers RNA binding. The predicted amino acid sequence of hnRNP U contains 806 amino acids (88,939 Daltons), and shows no extensive homology to any known proteins. The N-terminus is rich in acidic residues and the C-terminus is glycine-rich. In addition, a glutamine-rich stretch, a putative NTP binding site and a putative nuclear localization signal are present. It could not be defined from the sequence what segment of the protein confers its RNA binding activity. We identified an RNA binding activity within the C-terminal glycine-rich 112 amino acids. This region, designated U protein glycine-rich RNA binding region (U-gly), can by itself bind RNA. Furthermore, fusion of U-gly to a heterologous bacterial protein (maltose binding protein) converts this fusion protein into an RNA binding protein. A 26 amino acid peptide within U-gly is necessary for the RNA binding activity of the U protein. Interestingly, this peptide contains a cluster of RGG repeats with characteristic spacing and this motif is found also in several other RNA binding proteins. We have termed this region the RGG box and propose that it is an RNA binding motif and a predictor of RNA binding activity.     

Actin-binding domain of mouse plectin. Crystal structure and binding to vimentin.

Plectin, a large and widely expressed cytolinker protein, is composed of several subdomains that harbor binding sites for a variety of different interaction partners. A canonical actin-binding domain (ABD) comprising two calponin homology domains (CH1 and CH2) is located in proximity to its amino terminus. However, the ABD of plectin is unique among actin-binding proteins as it is expressed in the form of distinct, plectin isoform-specific versions. We have determined the three-dimensional structure of two distinct crystalline forms of one of its ABD versions (pleABD/2alpha) from mouse, to a resolution of 1.95 and 2.0 A. Comparison of pleABD/2alpha with the ABDs of fimbrin and utrophin revealed structural similarity between plectin and fimbrin, although the proteins share only low sequence identity. In fact, pleABD/2alpha has been found to have the same compact fold as the human plectin ABD and the fimbrin ABD, differing from the open conformation described for the ABDs of utrophin and dystrophin. Plectin harbors a specific binding site for intermediate filaments of various types within its carboxy-terminal R5 repeat domain. Our experiments revealed an additional vimentin-binding site of plectin, residing within the CH1 subdomain of its ABD. We show that vimentin binds to this site via the amino-terminal part of its rod domain. This additional amino-terminal intermediate filament protein binding site of plectin may have a function in intermediate filament dynamics and assembly, rather than in linking and stabilizing intermediate filament networks.     

Biogenesis of YidC Cytoplasmic Membrane Substrates Is Required for Positioning of Autotransporter IcsA at Future Poles

Localization of proteins to specific sites within bacterial cells is often critical to their function. In rod-shaped bacteria, proteins involved in diverse and important cell processes localize to the cell poles. The molecular mechanisms by which these proteins are targeted to the pole, however, are poorly understood. The Shigella autotransporter protein IcsA, which is localized to the pole on the surface of the bacterium, is targeted to the pole in the cytoplasm by a mechanism that is conserved across multiple Gram-negative bacterial species and has thus served as an important and informative model for studying polar localization. We present evidence that in Escherichia coli, the establishment of polar positional information recognized by IcsA requires the activity of the cytoplasmic membrane protein insertase YidC. We show that the role of YidC in IcsA localization is independent of the cell septation and cytokinesis proteins FtsQ and FtsEX. FtsQ is required for polar localization of IcsA and, based on cross-linking studies, is inserted in the vicinity of YidC, but, we find, is not dependent on YidC for membrane insertion. FtsEX is a YidC substrate, but we find that it is not required for polar localization of IcsA. These findings indicate that polar positional information recognized by IcsA depends on one or more membrane proteins that require YidC for proper membrane insertion.     

Biological activity of human immunodeficiency virus type 1 Vif requires membrane targeting by C-terminal basic domains.

Human immunodeficiency virus type 1 (HIV-1) encodes a Vif protein which is important for virus replication and infectivity. Vif is a cytoplasmic protein which exists in both membrane-associated and soluble forms. The membrane-associated form is an extrinsic membrane protein which is tightly associated with the cytoplasmic side of membranes. We have analyzed the mechanism of membrane targeting of Vif and its role in HIV-1 replication. Mutagenesis studies demonstrate that C-terminal basic domains are required for membrane association. Vif mutations which disrupt membrane association also inhibit HIV-1 replication, indicating that membrane localization of Vif is likely to be required for its biological activity in vivo. Membrane binding of Vif is almost completely abolished by trypsin treatment of membranes. These results demonstrate that membrane localization of Vif requires C-terminal basic domains and interaction with a membrane-associated protein(s). This interaction may serve to direct Vif to a specific cellular site, since immunofluorescence staining and plasma membrane fractionation studies show that Vif is localized predominantly to an internal cytoplasmic compartment rather than to the plasma membrane. The mechanism of membrane targeting of Vif is different in some respects from that of other extrinsic membrane proteins, such as Ras, Src, and MARCKS, which utilize a basic domain together with a lipid modification for membrane targeting. Membrane targeting of Vif is likely to play an important role in HIV-1 replication and thus may be a therapeutic target.