PubMed · 8924990
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F A Sagliocco, P A Moore, A J Brown. 1996. Polysome analysis.. https://doi.org/10.1385/0-89603-319-8%3A297
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Proteins terminating in the CAAX motif, for example Ras and the yeast a-factor mating pheromone, are prenylated, trimmed of their last three amino acids, and carboxyl-methylated. The enzymes that mediate these activities, collectively referred to as CAAX processing components, have been identified genetically in Saccharomyces cerevisiae. Whereas the Ram1p/Ram2p prenyltransferase is a cytosolic soluble enzyme, sequence analysis predicts that the other CAAX processing components, the Rce1p and Ste24p proteases and the Ste14p methyltransferase, contain multiple membrane spans. To determine the intracellular site(s) at which CAAX processing occurs, we have examined the localization of the CAAX proteases Rce1p and Ste24p by subcellular fractionation and indirect immunofluorescence. We find that both of these proteases are associated with the endoplasmic reticulum (ER) membrane. In addition to having a role in CAAX processing, the Ste24p protease catalyzes the first of two cleavage steps that remove the amino-terminal extension from the a-factor precursor, suggesting that the first amino-terminal processing step of a-factor maturation also occurs at the ER membrane. The ER localization of Ste24p is consistent with the presence of a carboxyl-terminal dilysine ER retrieval motif, although we find that mutation of this motif does not result in mislocalization of Ste24p. Because the ER is not the ultimate destination for a-factor or most CAAX proteins, our results imply that a mechanism must exist for the intracellular routing of CAAX proteins from the ER membrane to other cellular sites.
TFIID is a multiprotein complex comprised of the TATA-binding protein (TBP) and an array of TBP-associated factors (TAFIIs). Whereas TBP is sufficient for basal transcription in conjunction with other general transcription factors and RNA polymerase II, TAFIIs are additionally required for activator-dependent transcription in mammalian cell-free transcription systems. However, recent in vivo studies carried out in yeast suggest that TAFIIs are not globally required for activator function. The discrepancy between in vivo yeast studies and in vitro mammalian cell-free systems remains to be resolved. In this study, we describe a mammalian cell-free transcription system reconstituted with only recombinant proteins and epitope-tagged multiprotein complexes. Transcriptional activation can be recapitulated in this highly purified in vitro transcription system in the absence of TAFIIs. This TBP-mediated activation is not induced by human mediator, another transcriptional coactivator complex potentially implicated in activator response. In contrast, general transcription factors TFIIH and TFIIA play a significant role in TBP-mediated activation, which can be detected in vitro with Gal4 fusion proteins containing various transcriptional activation domains. Our data, therefore, suggest that TFIIH and TFIIA can mediate activator function in the absence of TAFIIs.
A cleanup method based on anion-exchange solid-phase extraction (SPE) was developed to render biological extracts suitable for the analysis of hexose phosphates with a modified anion-exchange chromatography method and pulsed amperometric detection. The method was applied to cell extracts of Saccharomyces cerevisiae obtained by using cold methanol as quenching agent and chloroform as extraction solvent. It was shown that pretreatment of the cell extract with SPE markedly improved the quality of the liquid chromatography analysis with recoveries of the sugar phosphates close to 100%. Furthermore, the method allowed for sample enrichment and the original extraction procedure could be simplified by implementing SPE early in the extraction protocol.