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In the accompanying paper [Adams, H., Scotti, P.A., de Cock, H., Luirink, J. & Tommassen, J. (2002) Eur. J. Biochem. 269, 5564-5571.], we showed that the precursor of outer-membrane protein PhoE of Escherichia coli with a Gly to Leu substitution at position -10 in the signal sequence (G-10L) is targeted to the SecYEG translocon via the signal-recognition particle (SRP) route, instead of via the SecB pathway. Here, we studied the fate of the mutant precursor in a prlA4 mutant strain. prlA mutations, located in the secY gene, have been isolated as suppressors that restore the export of precursors with defective signal sequences. Remarkably, the G-10L mutant precursor, which is normally exported in a wild-type strain, accumulated strongly in a prlA4 mutant strain. In vitro cross-linking experiments revealed that the precursor is correctly targeted to the prlA4 mutant translocon. However, translocation across the cytoplasmic membrane was defective, as appeared from proteinase K-accessibility experiments in pulse-labeled cells. Furthermore, the mutant precursor was found to accumulate when expressed in a secY40 mutant, which is defective in the insertion of integral-membrane proteins but not in protein translocation. Together, these data suggest that SecB and SRP substrates are differently processed at the SecYEG translocon.

This paper examines whether the in vivo behavior of yeast glycolysis can be understood in terms of the in vitro kinetic properties of the constituent enzymes. In nongrowing, anaerobic, compressed Saccharomyces cerevisiae the values of the kinetic parameters of most glycolytic enzymes were determined. For the other enzymes appropriate literature values were collected. By inserting these values into a kinetic model for glycolysis, fluxes and metabolites were calculated. Under the same conditions fluxes and metabolite levels were measured. In our first model, branch reactions were ignored. This model failed to reach the stable steady state that was observed in the experimental flux measurements. Introduction of branches towards trehalose, glycogen, glycerol and succinate did allow such a steady state. The predictions of this branched model were compared with the empirical behavior. Half of the enzymes matched their predicted flux in vivo within a factor of 2. For the other enzymes it was calculated what deviation between in vivo and in vitro kinetic characteristics could explain the discrepancy between in vitro rate and in vivo flux.

In the parallel paper, we developed a property to characterize drug efflux pumps, i.e. the reduced relative resistance (RRR). Using this RRR, we here investigate whether the observed diversity in human multidrug resistance (MDR) phenotypes might be due to variable levels of P-glycoprotein encoded by MDR1. We analyzed resistance phenotypes of various human cell lines in which either one, or both, classical human multidrug resistance genes, MDR1 and MDR3, are overexpressed. In addition, RRR values were calculated for MDR phenotypes presented in the literature. The results suggest that more than a single mechanism is required to account for the observed phenotypic diversity of classical multidrug resistance. This diversity is only partly due to differences in plasma membrane permeabilities between cell line families. It is discussed whether the alternative MDR phenotypes might be MDR1 phenotypes modified by other factors that do not themselves cause MDR. The method we here apply may also be useful for other nonspecific enzymes or pumps.