The novel protein Ccz1p required for vacuolar assembly in Saccharomyces cerevisiae functions in the same transport pathway as Ypt7p.

CCZ1 was previously identified by the sensitivity of ccz1(delta) mutants to high concentrations of Caffeine and the divalent ions Ca(2+ )and Zn(2+). In this paper we show that deletion of CCZ1 leads to aberrant vacuole morphology, similar to the one reported for the family of vacuolar protein sorting (vps) mutants of class B. The ccz1(&Dgr;) cells display severe vacuolar protein sorting defects for both the soluble carboxipeptidase Y and the membrane-bound alkaline phosphatase, which are delivered to the vacuole by distinct routes. Ccz1p is a membranous protein and the vast majority of Ccz1p resides in late endosomes. These results, along with a functional linkage found between the CCZ1 and YPT7 genes, indicate that the site of Ccz1p function is at the last step of fusion of multiple transport intermediates with the vacuole.

The F-box protein Rcy1p is involved in endocytic membrane traffic and recycling out of an early endosome in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, endocytic material is transported through different membrane-bound compartments before it reaches the vacuole. In a screen for mutants that affect membrane trafficking along the endocytic pathway, we have identified a novel mutant disrupted for the gene YJL204c that we have renamed RCY1 (recycling 1). Deletion of RCY1 leads to an early block in the endocytic pathway before the intersection with the vacuolar protein sorting pathway. Mutation of RCY1 leads to the accumulation of an enlarged compartment that contains the t-SNARE Tlg1p and lies close to areas of cell expansion. In addition, endocytic markers such as Ste2p and the fluorescent dyes, Lucifer yellow and FM4-64, were found in a similar enlarged compartment after their internalization. To determine whether rcy1Delta is defective for recycling, we have developed an assay that measures the recycling of previously internalized FM4-64. This method enables us to follow the recycling pathway in yeast in real time. Using this assay, it could be demonstrated that recycling of membranes is rapid in S. cerevisiae and that a major fraction of internalized FM4-64 is secreted back into the medium within a few minutes. The rcy1Delta mutant is strongly defective in recycling.

Electrochemical detection of nitric oxide production in perfused pig coronary artery: comparison of the performances of two electrochemical sensors.

In situ direct measurement of nitric oxide (NO) in biological media is now possible by means of electrochemical detection. In the literature, there are principally two amperometric approaches based on the direct oxidation of NO either on a sensor made from platinum/ iridium (Pt/Ir) alloy coated with a three-layered membrane or on a nickel porphyrin and Nafion-coated carbon fiber electrode. Nonetheless, the exact nature of the experimental amperometric signal obtained with the Pt/Ir system was never authenticated as being related to NO. This study compared responses of two sensors to the inhibition effect of Nomega-nitro-L-arginine (L-NA) as the amperometric signals produced by 5-hydroxytryptamine (5-HT) on isolated pig coronary preparations. These amperometric signals could be attributed to NO only for the nickel porphyrin and Nafion-coated carbon fiber electrode. Indeed, voltammetric characterization of the electrochemical response demonstrated only variations of the baseline current upon additions of either SNAP or NO on the Pt/Ir electrode instead of anodic peak current displayed at 0.63-0.75 V for the other system. Nitrites induced baseline current variations with the Pt/Ir electrode, similar to those obtained with S-nitroso-N-acetyl-dl-penicillamine (SNAP) or NO. This study highlights the potential hazards and pitfalls that may be associated with the use of a Pt/Ir sensor calibrated with SNAP solutions for the detection of NO production in various biological systems.