Compartmentalisation and localisation of the translation initiation factor (eIF) 4F complex in normally growing fibroblasts.

Previous observations of association of mRNAs and ribosomes with subcellular structures highlight the importance of localised translation. However, little is known regarding associations between eukaryotic translation initiation factors and cellular structures within the cytoplasm of normally growing cells. We have used detergent-based cellular fractionation coupled with immunofluorescence microscopy to investigate the subcellular localisation in NIH3T3 fibroblasts of the initiation factors involved in recruitment of mRNA for translation, focussing on eIF4E, the mRNA cap-binding protein, the scaffold protein eIF4GI and poly(A) binding protein (PABP). We find that these proteins exist mainly in a soluble cytosolic pool, with only a subfraction tightly associated with cellular structures. However, this "associated" fraction was enriched in active "eIF4F" complexes (eIF4E.eIF4G.eIF4A.PABP). Immunofluorescence analysis reveals both a diffuse and a perinuclear distribution of eIF4G, with the perinuclear staining pattern similar to that of the endoplasmic reticulum. eIF4E also shows both a diffuse staining pattern and a tighter perinuclear stain, partly coincident with vimentin intermediate filaments. All three proteins localise to the lamellipodia of migrating cells in close proximity to ribosomes, microtubules, microfilaments and focal adhesions, with eIF4G and eIF4E at the periphery showing a similar staining pattern to the focal adhesion protein vinculin.

Feedback control of polyketide metabolism during tylosin production.

Tylosin is produced by Streptomyces fradiae via a combination of polyketide metabolism and synthesis of three deoxyhexose sugars, of which mycaminose is the first to be added to the polyketide aglycone, tylactone (protylonolide). Previously, disruption of the gene (tylMII) encoding attachment of mycaminose to the aglycone unexpectedly abolished accumulation of the latter, raising the possibility of a link between polyketide metabolism and deoxyhexose biosynthesis in S. fradiae. However, at that time, it was not possible to eliminate an alternative explanation, namely, that downstream effects on the expression of other genes, not involved in mycaminose metabolism, might have contributed to this phenomenon. Here, it is shown that disruption of any of the four genes (tylMI--III and tylB) specifically involved in mycaminose biosynthesis elicits a similar response, confirming that production of mycaminosyl-tylactone directly influences polyketide metabolism in S. fradiae. Under similar conditions, when mycaminose biosynthesis was specifically blocked by gene disruption, accumulation of tylactone could be restored by exogenous addition of glycosylated tylosin precursors. Moreover, certain other macrolides, not of the tylosin pathway, were also found to elicit qualitatively similar effects. Comparison of the structures of stimulatory macrolides will facilitate studies of the stimulatory mechanism.