Macromolecular assemblies regulate nonvesicular phosphatidylserine traffic in yeast.

PtdSer (phosphatidylserine) is synthesized in the endoplasmic reticulum and the related MAM (mitochondria-associated membrane), and transported to the PtdSer decarboxylases, Pds1p in the mitochondria, and Psd2p in the Golgi. Genetic and biochemical analyses of PtdSer transport are now revealing the role of specific protein and lipid assemblies on different organelles that regulate non-vesicular PtdSer transport. The transport of PtdSer from MAM to mitochondria is regulated by at least three genes: MET30 (encoding a ubiquitin ligase), MET4 (encoding a transcription factor), and one or more unknown genes whose transcription is regulated by MET4. MET30-dependent ubiquitination is required for the MAM to function as a competent donor membrane and for the mitochondria to function as a competent acceptor membrane. Non-vesicular transport of PtdSer to the locus of Psd2p is under the control of at least three genes, STT4 [encoding Stt4p (phosphatidylinositol 4-kinase)], PSTB2 (encoding the lipid-binding protein PstB2p) and PSD2 (encoding Psd2p). Stt4p is proposed to produce a pool of PtdIns4P that is necessary for lipid transport. PstB2p and Psd2p must be present on the acceptor membrane for PtdSer transport to occur. Psd2p contains a C2 (Ca(2+) and phospholipid binding sequence) domain that is required for lipid transport. Reconstitution studies with chemically defined donor membranes demonstrate that membrane domains rich in the anionic lipids, PtdSer, PtdIns4P and phosphatidic acid function as the most efficient donors of PtdSer to Psd2p. The emerging view is that macromolecular complexes dependent on protein-protein and protein-lipid interactions form between donor and acceptor membranes and serve to dock the compartments and facilitate phospholipid transport.

Simplified Reverse Dot Blot Analyses for Detecting of ras Oncogene Mutations.

Background: Mutations in members of the ras gene family (H-ras, K-ras, and N-ras) have been identified in various human malignancies. A variety of techniques have been used to test for ras mutations. Methods and Results: A simplified reverse dot blot (RDB) assay was used in this study. Polymerase chain reaction products were hybridized to nitrocellulose membrane-fixed synthetic probes (20 nucleotides long) specific for codons 12, 13, and 61 of H-, K-, and N-ras mutations and their wild-type sequences. No special treatment or modification of the probes was necessary to obtain adequate results in overnight film exposure when the polymerase chain reaction was carried out using (32)P-end labeled primers. It was demonstrated that this simplified RDB assay can also be used with fluorescein-11-dUTP and a chemiluminescence detection system. The RDB assay is more reliable than the single-strand conformation polymorphism (SSCP) assay. By comparison, the SSCP assay is significantly less sensitive and less specific. It was confirmed with sequencing that 11 (12%) of 93 SSCP assays were false positive and 2 (2%) were false negative, whereas no false positive or false negative RDB assay was detected. The RDB assay also provides more additional detailed information about the specific point mutation and amino acid change, which may have clinical implications in some tumors. Conclusions: The RDB assay is very sensitive and able to detect mutations when the mutant allele is in 1% of the cells and can be used to detect minimal residual disease, particularly in some cases of leukemia and myelodysplasia.