Deoxycytidine deaminases APOBEC3G (A3G) and APOBEC3F (A3F) (members of the apolipoprotein B mRNA-editing catalytic polypeptide 3 family) have RNA-binding motifs, invade assembling human immunodeficiency virus (HIV-1), and hypermutate reverse transcripts. Antagonistically, HIV-1 viral infectivity factor degrades these enzymes. A3G is enzymatically inhibited by binding RNA within an unidentified large cytosolic ribonucleoprotein, implying that RNA degradation during reverse transcription may activate intravirion A3G at the necessary moment. We purified a biologically active tandem affinity-tagged A3G from human HEK293T cells. Mass spectrometry and coimmunoprecipitation from HEK293T and T lymphocyte extracts identified many RNAbinding proteins specifically associated with A3G and A3F, including poly(A)-binding proteins (PABPs), YB-1, Ro-La, RNA helicases, ribosomal proteins, and Staufen1. Most strikingly, nearly all A3G-associated proteins were known to bind exclusively or intermittently to translating and/or dormant mRNAs. Accordingly, A3G in HEK293T and T lymphocyte extracts was almost completely in A3G-mRNA-PABP complexes that shifted reversibly between polysomes and dormant pools in response to translational inhibitors. For example arsenite, which inhibits 5-cap-dependent translational initiation, shifted mRNA-A3G-PABP from polysomes into stress granules in a manner that was blocked and reversed by the elongation inhibitor cycloheximide. Immunofluorescence microscopy showed A3G-mRNA-PABP stress granules only partially overlapping with Staufen1. A3G coimmunoprecipitated HIV-1 RNA and many mRNAs. Ribonuclease released nearly all A3G-associated proteins, including A3G homo-oligomers and A3G-A3F hetero-oligomers, but the viral infectivity factor remained bound. Many proteins and RNAs associated with A3G are excluded from A3G-containing virions, implying that A3G competitively partitions into virions based on affinity for HIV-1 RNA.The viral infectivity factor (Vif) 4 encoded by human immunodeficiency virus type-1 (HIV-1) neutralizes a potent anti-HIV-1 defense system that occurs specifically in T lymphocytes and to a lesser degree in macrophages, thus explaining the efficient replication in T cells of wild-type HIV-1 but not HIV-1(⌬vif) that lacks a functional vif gene (1-4). This antiviral defense system involves the deoxycytidine deaminases APOBEC3G (A3G) and its paralog APOBEC3F (A3F) (members of the apolipoprotein B mRNA-editing catalytic polypeptide 3 family), each of which contains two RNA-binding motifs and incorporates into assembling HIV-1 capsids where they cause lethal dC-to-dU hypermutations in the single-stranded viral DNA that transiently forms during reverse transcription (1, 2, 4 -10). This single-stranded DNA, which has a minus sense, is synthesized using viral RNA as a template and is released from the DNA-RNA hybrid when the RNA is degraded by a ribonuclease H inherent in reverse transcriptase. Vif binds specifically to A3G and A3F and recruits a multisubunit ubiquitin ligase complex containing...

When stimulated strongly, a hair cell's mechanically sensitive hair bundle may consume ATP too rapidly for replenishment by diffusion. To provide a broad view of the bundle's protein complement, including those proteins participating in energy metabolism, we used shotgun mass spectrometry methods to identify proteins of purified chicken vestibular bundles. In addition to cytoskeletal proteins, proteins involved in Ca(2+) regulation, and stress-response proteins, many of the most abundant bundle proteins that were identified by mass spectrometry were involved in ATP synthesis. After beta-actin, the cytosolic brain isoform of creatine kinase was the next most abundant bundle protein; at approximately 0.5 mM, creatine kinase is capable of maintaining high ATP levels despite 1 mM/s ATP consumption by the plasma-membrane Ca(2+)-ATPase. Consistent with this critical role in hair bundle function, the creatine kinase circuit is essential for high-sensitivity hearing as demonstrated by hearing loss in creatine kinase knockout mice.

Escherichia coli glycerol kinase (GK) displays "half-of-the-sites" reactivity toward ATP and allosteric regulation by fructose 1, 6-bisphosphate (FBP), which has been shown to promote dimer-tetramer assembly and to inhibit only tetramers. To probe the role of tetramer assembly, a mutation (Ser58-->Trp) was designed to sterically block formation of the dimer-dimer interface near the FBP binding site [Ormo, M., Bystrom, C., and Remington, S. J. (1998) Biochemistry 37, 16565-16572]. The substitution did not substantially change the Michaelis constants or alter allosteric regulation of GK by a second effector, the phosphocarrier protein IIAGlc; however, it eliminated FBP inhibition. Crystal structures of GK in complex with different nontransferable ATP analogues and glycerol revealed an asymmetric dimer with one subunit adopting an open conformation and the other adopting the closed conformation found in previously determined structures. The conformational difference is produced by a approximately 6.0 degrees rigid-body rotation of the N-terminal domain with respect to the C-terminal domain, similar to that observed for hexokinase and actin, members of the same ATPase superfamily. Two of the ATP analogues bound in nonproductive conformations in both subunits. However, beta, gamma-difluoromethyleneadenosine 5'-triphosphate (AMP-PCF2P), a potent inhibitor of GK, bound nonproductively in the closed subunit and in a putative productive conformation in the open subunit, with the gamma-phosphate placed for in-line transfer to glycerol. This asymmetry is consistent with "half-of-the-sites" reactivity and suggests that the inhibition of GK by FBP is due to restriction of domain motion.

Methods for quantitative analysis of proteins by mass spectrometry have progressed dramatically. While isotope-dilution approaches using selected reaction monitoring of tryptic peptides (also known as bottom up) have become common, the potential to use narrow mass extraction of high-resolution mass spectra provides a compelling alternative. We investigated the relationships between instrument performance and data processing with the aim of determining whether this approach can lead to robust bioanalytical assays for proteins. Our approach utilized off-line sample preparation combined with online sample extraction coupled to HPLC with the effluent from the analytical column directed to a high-resolution, high-mass accuracy quadrupole time-of-flight (qTOF) mass spectrometer operated in full scan mode. Narrow mass extraction of a single isotope from IGF-1 in the 7+ charge state (m/z 1093.5209) was used to generate extracted ion chromatograms. We found that with appropriate attention to instrument performance and data processing, quantitative protein assays with good sensitivity, high selectivity, and excellent analytical performance can be developed.