Regulation of Protein Synthesis in the Plasmodial Phase of Physarum polycephalum

Abstract

1. The rate of protein synthesis changes very little during the first 2-3 h (S phase) of the nuclear division cycle in plasmodia of Physarumpolycephalum and then increases continuously during G 2 phase, so that by the end of the cycle the rate has doubled relative to that in S phase. Protein synthesis appears to continue during mitosis.2. Fractionation of extracts of plasmodia, labelled with [3H]lysine for 1 h, by two-dimensional electrophoresis indicated that most if not all proteins are synthesised throughout the nuclear division cycle. However, two metabolically stable polypeptides, the synthesis of which occurs predominantly in G 2 phase, were detected.3. Using a double-labelling procedure, the differential rates of synthesis of 30 relatively abundant polypeptides were measured in relation to the nuclear division cycle. As a group, their differential rates of synthesis increase during the cycle so that their actual rates of synthesis increase 4-6-fold. This implies that their synthesis is regulated over and above any simple change due to a doubling in the number of genes during S phase.Growth of the plasmodial phase of the myxomycete, Pl~ysurum polycephalum, occurs without cell division, and the nuclei in the common cytoplasm divide with a high degree of synchrony [l]. There is no detectable delay (or GI phase) between the completion of nuclear division and S phase, which lasts 2 -3 h [2,3]. The remainder of the cycle (6 -7 h) is occupied by G2 phase; mitosis itself only lasts 20-30 min.The natural synchrony characteristic of the plasmodial phase of Physarum makes it a useful organism for the study of biochemical processcs in relation to nuclear division. The synthesis of protein between successive nuclear divisions has previously been determined by pulse-labelling with ["S]-methionine [4] and by an indirect reference to the synthesis of total nucleic acid [5]. However, unequivocal interpretation of pulse-labelling experiments is difficult, and the number of measurements required in the other study led to considerable experimental error in the values calculated for the increment in protein.Initially it was considered [5] that the isotope dilution method, developed for the study of metabolically stable nucleic acids [3], would not be directly applicable to the analysis of protein synthesis, because of the largely unknown extent of protein turnover. Further reflection suggested that amino acid labelling could be used provided that sufficient growth in tinlabelled medium was allowed before beginning specific activity measurements. If the chase period is sufficiently long, then radioactivity in proteins with a short half-life will be progrcssively lost and will accumulate in proteins that are either completely stable during growth or have a lifetime that is long by comparison with the intermitotic period. Once radioactivity is confined to metabolically stable proteins, the decrease with time of the specific activity of the total protein of the cell will be a simple index of net synthesis.In this paper we present...

Specific ribosomal binding sites for aminoglycoside antibiotics in vivo: Their role in mistranslation and inhibition of protein synthesis

“…Application of these latter figures to in vivo protein synthesis generally could certainly account for production of completely non-functional proteins (which are rapidly degraded [21]) by ribosomes with the antibiotics tightly bound to their specific sites. Our results further support the suggestion that such ribosomes do not recover normal function [ 1,3,5]. Gradual decline and eventual absence of unaffected ribosomes in treated cultures of sensitive organisms could then account for the irreversible inhibition of growth and the bactericidal effects.…”

“…Furthermore, the Final titre of approx. 1 molecule of antibiotic per 30S ribosome, when growth inhibition was complete, accorded with the suggestion of specific inhibitory binding sites on 30S ribosomes [4,5,7]. Tight binding of streptomycin (and other aminoglycoside antibiotics) to sensitive ribosomes in vitro has also been variously reported [8][9][10].…”

“…Binding of high affinity -so as to be practically irreversible -between streptomycin or dihydrostrep. tomycin and sensitive ribosomes in vivo has been suggested as an essential component of the antibiotic effect, namely irreversible inhibition of growth [ 1,5]. This was based on studies !n rive of Escherichia coli cultures treated with these afatibiotics.…”

Patterns of tubulin isotype synthesis and usage during mitotic spindle morphogenesis in Physarum

“…The plasmodial tubulins have been shown to be relatively stable polypeptides and may persist throughout several cell cycles [Laffler et al, 1981;Turnock et al, 1981 1; however, whether this chemical longevity reflects biological function and usage is not known. This question is particularly pertinent in a system where the tubulins are synthesized periodically.…”

An immunological approach to enrich a mitotic stimulator and to reveal G2-phase-specific proteins in Physarum polycephalum.

“…No protein synthesized at different rates during parts of the cell cycle could be identified. In several eucaryotic organisms, like Saccharomyces cerevisiae (6), Physarum polycephalum (4,9,21) or HeLa cells (3,14), certain proteins are synthesized at different rates during the cell cycle. Among the few variable proteins, tubulins have been identified in HeLa cells (3) as well as in Physarum polycephalum (4,9).Apart from periodic synthesis through the cell cycle, the question remains whether there are specific proteins that are only present at a distinct phase of the cell cycle.…”