A systematic study of yeast sterol biosynthetic protein-protein interactions using the split-ubiquitin system.
ABSTRACT Sterol biosynthesis occurs in the ER and most sterol biosynthetic enzymes have transmembrane domains. However, due to difficulties in characterizing membrane protein-protein interactions, the nature of the sterol biosynthetic complex as well as in vivo interactions between various enzymes have not been described. We employed a split-ubiquitin membrane protein yeast two-hybrid system to characterize interactions between sterol biosynthetic proteins. Fourteen bait constructs were co-transformed into a reporter yeast strain with 14 prey constructs representing all sterol enzymatic reactions beginning with the synthesis of squalene. Our results not only confirmed several previous interactions, but also allowed us to identify novel interactions. Based on these results, ergosterol biosynthetic enzymes display specific protein-protein interactions forming a functional complex we designate, the ergosome. In this complex, Erg11p, Erg25p, Erg27p, and Erg28p appear to form a core center that can interact with other enzymes in the pathway. Also Erg24p and Erg2p, two enzymes that are sensitive to morpholine antifungals, appear to interact with one another; however, the profile of protein interaction partners appears to be unique. Erg2p and Erg3p, two enzymes catalyzing sequential reactions also appear to have different interaction partners. Our results provide a working model as to how sterol biosynthetic enzymes are topologically organized not only in yeast but in plant and animal systems that share many of these biosynthetic reactions.
- SourceAvailable from: Brian C Monk[Show abstract] [Hide abstract]
ABSTRACT: Bitopic integral membrane proteins with a single transmembrane helix play diverse roles in catalysis, cell signaling, and morphogenesis. Complete monospanning protein structures are needed to show how interaction between the transmembrane helix and catalytic domain might influence association with the membrane and function. We report crystal structures of full-length Saccharomyces cerevisiae lanosterol 14α-demethylase, a membrane monospanning cytochrome P450 of the CYP51 family that catalyzes the first postcyclization step in ergosterol biosynthesis and is inhibited by triazole drugs. The structures reveal a well-ordered N-terminal amphipathic helix preceding a putative transmembrane helix that would constrain the catalytic domain orientation to lie partly in the lipid bilayer. The structures locate the substrate lanosterol, identify putative substrate and product channels, and reveal constrained interactions with triazole antifungal drugs that are important for drug design and understanding drug resistance.Proceedings of the National Academy of Sciences 02/2014; DOI:10.1073/pnas.1324245111 · 9.81 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Sterols are crucial components of eukaryotic membranes that control membrane fluidity and permeability. They play an important role in cell signaling, polarity and sorting. Since many steps in the pathway are essential, sterol biosynthesis inhibitors (SBI) are widely used as antifungal agents. This work reports the identification and the characterization of a C-4 sterol methyl oxidase (SMO), the first gene involved in the sterol biosynthetic pathway, so far described from an arbuscular mycorrhizal fungus. The sequence, called GintSMO, shows a primary structure, a hydrophobicity profile and a pattern of histidine-rich motifs which are typical of C-4 methyl sterol oxidases. The complementation assay in a Saccharomyces cerevisiae mutant strain demonstrates that GintSMO encodes a functional SMO. Changes in GintSMO transcript levels and in the amount of the sterol precursor squalene were observed in in vitro grown extraradical structures exposed to the fenpropimorph SBI fungicide.Fungal Genetics and Biology 04/2009; 46(6-7):486-95. DOI:10.1016/j.fgb.2009.03.002 · 3.26 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Low dielectric constant (k) materials applied for the ULSI interconnect insulator are required to meet the fast development of high-speed devices. Introducing nanopores into dielectrics reduces their k value effectively but also degrades the film hardness drastically. In this work, Young's moduli of several promising porous low-k films are determined by an improved technique of surface acoustic waves (SAWs). The twin-transducer is introduced to insure the propagation alignment of the laser generated SAWs, consequently increasing the accuracy of the measurement.Solid-State and Integrated Circuits Technology, 2004. Proceedings. 7th International Conference on; 11/2004