Subcellular localization of Ras proteins to the plasma membrane is accomplished in part by covalent attachment of a farnesyl moiety to the conserved CaaX box cysteine.Farnesylation targets Ras to the endoplasmic reticulum (ER), where additional processing steps occur, resulting in translocation of Ras to the plasma membrane.The mechanism(s) by which this occurs is not well understood.In this report, we show that plasma membrane localization of Ras2p in Saccharomyces cerevisiae does not require the classical secretory pathway or a functional Golgi apparatus.However, when the classical secretory pathway is disrupted, plasma membrane localization requires Erf2p, a protein that resides in the ER membrane and is required for efficient palmitoylation of Ras2p.Deletion of ERF2 results in a Ras2p steady-state localization defect that is more severe when combined with sec-ts mutants or brefeldin A treatment.The Erf2p-dependent localization of Ras2p correlates with the palmitoylation of Cys-318.An Erf2p-Erf4p complex has recently been shown to be an ER-associated palmitoyltransferase that can palmitoylate Cys-318 of Ras2p (S.Lobo, W. K. Greentree, M. E. Linder, and R. J. Deschenes, J. Biol.Chem.277:41268-41273, 2002).Erf2-dependent palmitoylation as well as localization of Ras2p requires a region of the hypervariable domain adjacent to the CaaX box.These results provide evidence for the existence of a palmitoylation-dependent, nonclassical endomembrane trafficking system for the plasma membrane localization of Ras proteins.
Photoreceptor dysplasia, characterized by formation of folds and (pseudo-)rosettes in the outer retina, is associated with loss of functional nuclear receptor subfamily 2 group E member 3 (NR2E3) and neural retina leucine-zipper (NRL) in both humans and mice. A sensitized chemical mutagenesis study to identify genetic modifiers that suppress photoreceptor dysplasia in Nr2e3rd7mutant mice identified line Tvrm222, which exhibits a normal fundus appearance in the presence of the rd7 mutation. The Tvrm222 modifier of Nr2e3rd7/rd7 was localized to Chromosome 6 and identified as a missense mutation in the FERM domain containing 4B (Frmd4b) gene. The variant is predicted to cause the substitution of a serine residue 938 with proline (S938P). The Frmd4bTvrm222 allele was also found to suppress outer nuclear layer (ONL) rosettes in Nrl-/- mice. Fragmentation of the external limiting membrane (ELM), normally observed in rd7 and Nrl-/-mouse retinas, was absent in the presence of the Frmd4bTvrm222 allele. FRMD4B, a binding partner of cytohesin 3, is proposed to participate in cell junction remodeling. Its biological function in photoreceptor dysplasia has not been previously examined. In vitro experiments showed that the FRMD4B938P variant fails to be efficiently recruited to the cell surface upon insulin stimulation. In addition, we found a reduction in protein kinase B phosphorylation and increased levels of cell junction proteins, Catenin beta 1 and tight junction protein 1, associated with the cell membrane in Tvrm222 retinas. Taken together, this study reveals a critical role of FRMD4B in maintaining ELM integrity and in rescuing morphological abnormalities of the ONL in photoreceptor dysplasia.
Verticillium dahliae is highly prone to pathogenic differentiation and influenced by host cotton's resistance. To better understand the mechanisms of this phenomenon, we applied the host selective pressures of resistant and susceptible cotton varieties on V. dahliae strain Vd076 within an artificial cotton Verticillium wilt nursery and greenhouse. Consequently, among the offspring strains, high virulence strains exhibited higher levels of physiological characteristics and genetic diversity compared to moderate and low virulence strains. Moreover, whole genome resequencing revealed that the Ka/Ks ratio of single nucleotide polymorphism (SNPs) in the majority of the offspring strains was about 0.6, indicating an adverse selection impact in the offspring strains. Pathogenicity assays demonstrated that the virulence of the offspring strains triggered by continuous induction of disease-resistant cotton cultivar increased from the 4th generation and reached its peak by the 6th generation. Additionally, the transcriptome analysis revealed that the 4th and 6th generations of strains differentially expressed genes (DEGs) accumulated a significant number of response genes associated with pathogen pathogenicity differentiation, including the mitogen-activated protein kinase (MAPK) signaling pathway, amino and antibiotic biosynthesis, phenylpropanoid metabolism. Furthermore, VDAG_04757, VDAG_06462, VDAG_03218, and VDAG_08487 genes exhibited significant correlation with the pathogenicity of V. dahliae. Collectively, this study has significant implications for elucidating the evolution of virulence in V. dahliae induced by the host, as well as for advancing holistic strategies for preventing and managing Verticillium wilt disease.
Ras oncogene proteins are plasma membrane-associated signal transducers that are found in all eukaryotes. Posttranslational addition of lipid to a carboxyl-terminal CaaX box (where "C" represents a cysteine, "a" is generally an aliphatic residue, andX can be any amino acid) is required to target Ras proteins to the cytosolic surface of the plasma membrane. The pathway by which Ras translocates from the endoplasmic reticulum to the plasma membrane is currently not clear. We have performed a genetic screen to identify components of the Ras plasma membrane localization pathway. Mutations in two genes, ERF2 and ERF4/SHR5, have been shown to affect the palmitoylation and subcellular localization of Ras proteins. In this report, we show that Erf4p is localized on the endoplasmic reticulum as a peripheral membrane protein in a complex with Erf2p, an integral membrane protein that was identified from the same genetic screen. Erf2p has been shown to be required for the plasma membrane localization of GFP-Ras2p via a pathway distinct from the classical secretory pathway (X. Dong and R. J. Deschenes, manuscript in preparation). We show here that Erf4p, like Erf2p, is involved in the plasma membrane localization of Ras2p. Erf2p and Erf4p represent components of a previously uncharacterized subcellular transport pathway involved in the plasma membrane targeting of Ras proteins.
Sphingolipids, lipids with a common sphingoid base (also termed long chain base) backbone, play essential cellular structural and signaling functions. Alterations of sphingolipid levels have been implicated in many diseases, including neurodegenerative disorders. However, it remains largely unclear whether sphingolipid changes in these diseases are pathological events or homeostatic responses. Furthermore, how changes in sphingolipid homeostasis shape the progression of aging and neurodegeneration remains to be clarified. We identified two mouse strains, flincher (fln) and toppler (to), with spontaneous recessive mutations that cause cerebellar ataxia and Purkinje cell degeneration. Positional cloning demonstrated that these mutations reside in the Lass1 gene. Lass1 encodes (dihydro)ceramide synthase 1 (CerS1), which is highly expressed in neurons. Both fln and to mutations caused complete loss of CerS1 catalytic activity, which resulted in a reduction in sphingolipid biosynthesis in the brain and dramatic changes in steady-state levels of sphingolipids and sphingoid bases. In addition to Purkinje cell death, deficiency of CerS1 function also induced accumulation of lipofuscin with ubiquitylated proteins in many brain regions. Our results demonstrate clearly that ceramide biosynthesis deficiency can cause neurodegeneration and suggest a novel mechanism of lipofuscin formation, a common phenomenon that occurs during normal aging and in some neurodegenerative diseases.
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