A high-yield cell-free system of protein synthesis of mouse liver

1. A fractionated cell-free system of protein synthesis has been developed from mouse liver. It is composed of polysomes, "pH 5" fraction, Mg2+, K+, ATP and a ATP generating system. 2. It operates optimally at 30-37 degrees C, in the presence of 4 mM MgCl2 and 90 mM KCl. 3. Spermine is highly inhibitory, while spermidine shows a bimodal action, in that submillimolar concentrations stimulate, while millimolar concentrations inhibit protein synthesis. 4. Both spermine and spermidine show an interesting selectivity, in that, even though they inhibit incorporation of amino acids into most proteins, they stimulate incorporation into a few proteins. 5. The system can be rendered mRNA-dependent, either by preincubation or by treatment with micrococcal nuclease. In both cases globin mRNA as well as TMV RNA are faithfully translated. 6. Compared to other published mammalian fractionated cell-free systems, the mouse liver system is more efficient by approximately one order of magnitude, since the rate of incorporation of leucine per min is 30 pmol/mg protein or 435 pmol/mg RNA or 1 mol/mol ribosomes.     

RNA association or phosphorylation of the RS domain prevents aggregation of RS domain-containing proteins

Domains rich in alternating arginine and serine residues (RS domains) are found in a large number of eukaryotic proteins involved in several cellular processes. According to the prevailing view RS domains function as protein interaction domains, thereby promoting the assembly of higher-order cellular structures. Furthermore, recent data demonstrated that the RS regions of several SR splicing factors directly contact the pre-mRNA in a nonsequence specific but functionally important fashion. Using a variety of biochemical approaches, we now demonstrate that the RS domains of three proteins, not directly associated with the splicing reaction, such as lamin b receptor, acinus and peroxisome proliferator-activated receptor gamma coactivator-1 alpha, associate mainly with nuclear RNA and that this association is conducive in retaining the proteins in a soluble form. Phosphorylation by SRPK1 prevents RNA association, yet it greatly increases the fraction of the proteins recovered in soluble form, thereby mimicking the RNA effect. Based on these results we propose that the tendency to self-associate and form aggregates is a general property of RS domain-containing proteins and could be attributed to their disordered structure. RNA binding or SRPK1-mediated phosphorylation prevents aggregation and may serve to modulate the RS domain interaction modes.     

Protein kinase CK2 phosphorylates and activates the SR protein-specific kinase 1

The serine/arginine subfamily of protein kinases has been conserved throughout evolution and its members are thought to play important roles in the regulation of multiple cellular processes. Mammalian SRPK1 has been considered as a constitutively active kinase that is predominantly expressed in testis. In the present study, recombinant GST-SRPK1 was used as substrate to identify potential protein kinase(s) in testis extracts, involved in phosphorylating and thereby regulating the activity of this enzyme. Using a panel of chromatography media, inhibition by heparin, immunoblot analysis, and phosphopeptide mapping, CK2 was determined to be the major kinase that phosphorylates SRPK1. Phosphorylation of SRPK1 by CK2 occurred mainly at Ser(51) and Ser(555) in vitro, and resulted in approximately 6-fold activation of the enzyme. These findings suggest that SRPK1 may be an important cellular target for CK2 action.     

Protein prenylation in Schizosaccharomyces pombe.

S. pombe is shown to be a powerful system for studies concerning attachment of polyisoprenoid moieties to proteins, due to its ability to take up exogenous mevalonic acid efficiently. The fission yeast can take up about 5% of the exogenously added mevalonic acid and incorporate approximately 10% of this into protein. By contrast, the uptake obtained with the budding yeast S. cerevisiae is less than 0.5%. HPLC analysis of total S. pombe protein-bound isoprenoids revealed that approximately 55% of the counts co-migrated with the geranylgeraniol standard, while approximately 45% of the counts co-migrated with farnesol. We could not detect any effects of mevinolin or other HMG-CoA reductase inhibitors in S. pombe.     

Inhibition of protein synthesis by acetyl-coenzyme A in a cell-free system: possible involvement of protein acetylation in the regulation of translation

Acetyl-coenzyme A (CoASAc) inhibits the rate of incorporation of amino acid into protein in a cell-free system of mouse liver. The effect is more pronounced when exogenous mRNA (tobacco mosaic virus or globin mRNA) rather than endogenous messages are used. Micromolar concentrations of the cofactor block initiation, while millimolar concentrations cause a more general inhibition of the translation process, that affects, in addition, the elongation step. Inclusion of [1-14C]acetyl-CoA in a protein synthesis reaction mixture results in a very rapid and selective labelling of a protein of 200 kd of the 'pH 5' fraction. The possible involvement of the acetylating event in the regulation of protein synthesis is discussed.