A series of diazonium salts bearing different para substituents was used to functionalize glassy carbon (GC) and pyrolyzed photoresist film (PPF) under electrografting conditions in the absence and presence of the radical scavenger diphenyl-1-picrylhydrazyl (DPPH). Depositions were monitored by electrochemical quartz crystal microbalance (EQCM) and the grafted layers were analyzed by atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS). DPPH was used to selectively suppress film growth by radical coupling and thereby to reveal the existence of secondary mechanisms involved in the polymerization. Differences in grafting behaviors between various diazonium ion derivatives can be explained by the influence of the para substituent's electronic properties on the electrophilic aromatic substitutions of diazonium ions on already grafted aromatic groups.
†These authors contributed equally to this study.We have studied the intracellular trafficking of Sit1 [ferrioxamine B (FOB) transporter] and Enb1 (enterobactin transporter) in Saccharomyces cerevisiae using green fluorescent protein (GFP) fusion proteins. Enb1 was constitutively targeted to the plasma membrane. Sit1 was essentially targeted to the vacuolar degradation pathway when synthesized in the absence of substrate. Massive plasma membrane sorting of Sit1 was induced by various siderophore substrates of Sit1, and by coprogen, which is not a substrate of Sit1. Thus, different siderophore transporters use different regulated trafficking processes. We also studied the fate of Sit1-mediated internalized siderophores. Ferrioxamine B was recovered in isolated vacuolar fractions, where it could be detected spectrophotometrically. Ferrioxamine B coupled to an inhibitor of mitochondrial protoporphyrinogen oxidase (acifluorfen) could not reach its target unless the cells were disrupted, confirming the tight compartmentalization of siderophores within cells. Ferrioxamine B coupled to a fluorescent moiety, FOB-nitrobenz-2-oxa-1,3-diazole, used as a Sit1-dependent iron source, accumulated in the vacuolar lumen even in mutants displaying a steady-state accumulation of Sit1 at the plasma membrane or in endosomal compartments. Thus, the fates of siderophore transporters and siderophores diverge early in the trafficking process.
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