Identification of proteins whose synthesis is modulated during the cell cycle of Saccharomyces cerevisiae

We examined the synthesis and turnover of individual proteins in the Saccharomyces cerevisiae cell cycle. Proteins were pulse-labeled with radioactive isotope (35S or 14C) in cells at discrete cycle stages and then resolved on two-dimensional gels and analyzed by a semiautomatic procedure for quantitating gel electropherogram-autoradiographs. The cells were obtained by one of three methods: (i) isolation of synchronous subpopulations of growing cells by zonal centrifugation.; (ii) fractionation of pulse-labeled steady-state cultures according to cell age; and (iii) synchronization of cells with the mating pheromone, alpha-factor. In confirmation of previous studies, we found that the histones H4, H2A, and H2B were synthesized almost exclusively in the late G1 and early S phases. In addition, we identified eight proteins whose rates of synthesis were modulated in the cell cycle, and nine proteins (of which five, which may well be related, were unstable, with half-lives of 10 to 15 min) that might be regulated in the cell cycle by periodic synthesis, modification, or degradation. Based on the time of maximal labeling in the cell cycle and on experiments with alpha-factor and hydroxyurea, we assigned the cell cycle proteins to two classes: proteins in class I were labeled principally in early G1 phase and at a late stage of the cycle, whereas those in class II were primarily synthesized at times ranging from late G1 to mid S phase. At least one major control point for the cell cycle proteins occurred between "start" and early S phase. A set of stress-responsive proteins was also identified and analyzed. The rates of synthesis of these proteins were affected by certain perturbations that resulted during selection of synchronous cell populations and by heat shock.     

Enhanced differential synthesis of proteins in a mammalian cell-free system by addition of polyamines.

Addition of the polyamines spermidine, spermine, or putrescine to a fractionated mammalian cell-free protein-synthesis system programmed by a variety of mRNAs results in a 3- to 5-fold stimulation of amino acid incorporation over that found in the absence of added polyamine. The mRNAs used as template were adenovirus mRNA, globin 9s mRNA, and RNA from the bacteriophages R17, Qbeta, and MS2. The relative amounts of 10 adenovirus polypeptides synthesized in vitro are altered by the addition of polyamines to the translation system to reflect more closely the relative amounts of these polypeptides synthesized in vivo. This qualititive improvement in translation products on addition of polyamines allow the analysis of a number of products which are at best only marginally synthesized in the absence of added polyamines. The low level of synthesis due to endogenous mRNA is stimulated by spermidine and spermine but a lesser extent by putrescine.     

mTORC1-independent translation control in mammalian cells by methionine adenosyltransferase 2A and S-adenosylmethionine

Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylmethionine (SAM). As the sole methyl-donor for methylation of DNA, RNA, and proteins, SAM levels affect gene expression by changing methylation patterns. Expression of MAT2A, the catalytic subunit of isozyme MAT2, is positively correlated with proliferation of cancer cells; however, how MAT2A promotes cell proliferation is largely unknown. Given that the protein synthesis is induced in proliferating cells and that RNA and protein components of translation machinery are methylated, we tested here whether MAT2 and SAM are coupled with protein synthesis. By measuring ongoing protein translation via puromycin labeling, we revealed that MAT2A depletion or chemical inhibition reduced protein synthesis in HeLa and Hepa1 cells. Furthermore, overexpression of MAT2A enhanced protein synthesis, indicating that SAM is limiting under normal culture conditions. In addition, MAT2 inhibition did not accompany reduction in mechanistic target of rapamycin complex 1 activity but nevertheless reduced polysome formation. Polysome-bound RNA sequencing revealed that MAT2 inhibition decreased translation efficiency of some fraction of mRNAs. MAT2A was also found to interact with the proteins involved in rRNA processing and ribosome biogenesis; depletion or inhibition of MAT2 reduced 18S rRNA processing. Finally, quantitative mass spectrometry revealed that some translation factors were dynamically methylated in response to the activity of MAT2A. These observations suggest that cells possess an mTOR-independent regulatory mechanism that tunes translation in response to the levels of SAM. Such a system may acclimate cells for survival when SAM synthesis is reduced, whereas it may support proliferation when SAM is sufficient.     

Identification of multiple proteins whose synthetic rates are enhanced by high amino acid levels in rat hepatocytes

Amino acids are key regulators of protein synthesis in liver. However, it remains to be determined whether amino acids stimulate synthesis of all or certain specific liver proteins. No techniques are currently available to simultaneously measure synthetic rates of several individual proteins. Here we report studies performed on rat hepatocyte primary cultures in which we used metabolic labeling with [(14)C]leucine, two-dimensional gel electrophoresis (2DGE), and tandem mass spectrometry to identify proteins that showed increased leucine incorporation when high amino acid levels were present in the media. Rat hepatocytes were isolated by in situ collagenase perfusion, cultured in serum-free medium containing insulin, and incubated for 2, 4, and 8 h in media of standard and high amino acid concentrations. SDS-PAGE and 2DGE were performed to separate proteins from cell lysates. Proteins that consistently showed increased synthesis on triplicate cultures, as detected by phosphorimaging of gels, were identified by tandem mass spectrometry. The combination of these approaches enabled the detection of 16 specific liver proteins whose synthetic rates were enhanced by increased amino acid concentration. These proteins are involved in specific functions such as translation initiation, protein folding and modification, oxidative phosphorylation, antioxidant defense, signal transduction, and transport, as well as cell motility and tissue integrity. No quantitative changes for any of these proteins were detected by gel staining, indicating that no detectable changes in protein concentration occurred. In contrast, measurable changes in synthetic rates occurred in 16 proteins. In conclusion, amino acids stimulate the synthesis of several liver proteins with important cellular functions.     

Identification of chikungunya virus interacting proteins in mammalian cells

Identification and characterization of virus host interactions is an essential step for the development of novel antiviral strategies. Very few studies have been targeted towards identification of chikungunya virus (CHIKV) interacting host proteins. In current study, virus overlay protein binding assay (VOPBA) and matrix-assisted laser desorption/ionization time of flight analysis (MALDI TOF/TOF) were employed for the identification of CHIKV binding proteins in mammalian cells. HSP70 and actin were identified as virus binding proteins in HEK-293T and Vero-E6 cells, whereas STAT-2 was identified as an additional protein in Vero-E6 cells. Pre-incubation with anti-HSP70 antibody and miRNA silencing of HSP70 significantly reduced the CHIKV production in HEK-293T and Vero-E6 cells at early time points. These results suggest that CHIKV exploits the housekeeping molecules such as actin, HSP70 and STAT-2 to establish infection in the mammalian cells.     

Identification of two testosterone-responsive testicular proteins in Sertoli cell-enriched culture medium whose secretion is suppressed by cells of the intact seminiferous tubule

Of 30 proteins identified in medium from primary Sertoli cell-enriched cultures, five of these proteins appear to increase primarily in response to testosterone. Using high performance liquid chromatography, two of these proteins, designated CMB-22 and CMB-23, were purified to apparent homogeneity from medium. Both are monomeric proteins with apparent molecular weights of Mr 37,000 and Mr 40,000, respectively. Both interact with concanavalin A and wheat germ agglutinin on lectin blots. CMB-22 has a pI of 5.8; CMB-23 has two distinctive isoelectric variants with pIs of 5.4 and 5.2, the latter variant was designated CMB-23 Isoform. Polyvalent antisera raised against purified CMB-22 and CMB-23 in rabbits cross-reacted with one another. Removal of carbohydrate from these proteins by either enzymatic or chemical treatments reduced their apparent molecular weights but did not abolish their size differences on sodium dodecyl sulfate-polyacrylamide gels. Peptide maps generated by Staphylococcus aureus protease V8 and visualized by silver staining and immunoblots suggest that CMB-22 and CMB-23 are similar but distinctive proteins that share almost identical epitopes. A radioimmunoassay was developed and used to measure total immunoreactive CMB-22-like material (CMB-22 plus CMB-23 immunoreactivity) in culture media, biological fluids, and tissue extracts. The results of these studies showed that the secretion of CMB-22-like material is unique with regard to other proteins that have been identified in Sertoli cell-enriched cultures. That is, CMB-22-like material is secreted by Sertoli cell-enriched cultures, but cannot be detected in media from cultures of intact tubular segments in vitro. In addition, immunoreactive material is also not detected in the testicular fluids from interstitium, tubule, or rete testis. This is in striking contrast to other Sertoli cell proteins which are present in substantial concentrations in these fluids. These observations suggest that the secretion and possibly the hormone responsiveness of CMB-22 and CMB-23 are normally suppressed in the intact tubule both in vivo and in vitro. We propose that studies of CMB-22 and CMB-23 will provide important insights into cell-cell interactions in the seminiferous epithelium.