Palmitoylation and Plasma Membrane Localization of Ras2p by a Nonclassical Trafficking Pathway in Saccharomyces cerevisiae
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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.Keywords:
Palmitoylation
Brefeldin A
Endomembrane system
Transport protein
Brefeldin A
Endomembrane system
Daucus carota
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In plant cells, as in animal cells, many macromolecules and membranes are transported by vesicle vectors through both the exocytotic and endocytotic pathways. In order to elucidate the mechanisms and molecular events of such trafficking we are using a set of drugs known to perturb membrane flow in plant cells in combination with immunocytochemical studies using a bank of monoclonal antibodies to various components of the endomembrane system and cell surface. In animal cells, one such drug, Brefeldin A, a fungal fatty acid derivative which causes disruption of the Golgi apparatus, has recently been used as a tool to dissect the mechanisms of vesicle flow from the endoplasmic reticulum to the Golgi apparatus and down the cisternae of the Golgi stack (1). It has been demonstrated that BFA also has a dramatic effect on the Golgi apparatus in higher plant cells (2,3,4). In this paper we report on recent work on the disruption of the plant Golgi apparatus with BFA and the redistribution of endomembrane marker epitopes after drug treatment of roots and suspension culture cells.
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Small GTP-binding proteins from the ADP-ribosylation factor (ARF) family are important regulators of vesicle formation and cellular trafficking in all eukaryotes. ARF activation is accomplished by a protein family of guanine nucleotide exchange factors (GEFs) that contain a conserved catalytic Sec7 domain. Here, we identified and characterized Secdin, a small-molecule inhibitor of Arabidopsis thaliana ARF-GEFs. Secdin application caused aberrant retention of plasma membrane (PM) proteins in late endosomal compartments, enhanced vacuolar degradation, impaired protein recycling, and delayed secretion and endocytosis. Combined treatments with Secdin and the known ARF-GEF inhibitor Brefeldin A (BFA) prevented the BFA-induced PM stabilization of the ARF-GEF GNOM, impaired its translocation from the Golgi to the trans-Golgi network/early endosomes, and led to the formation of hybrid endomembrane compartments reminiscent of those in ARF-GEF-deficient mutants. Drug affinity-responsive target stability assays revealed that Secdin, unlike BFA, targeted all examined Arabidopsis ARF-GEFs, but that the interaction was probably not mediated by the Sec7 domain because Secdin did not interfere with the Sec7 domain-mediated ARF activation. These results show that Secdin and BFA affect their protein targets through distinct mechanisms, in turn showing the usefulness of Secdin in studies in which ARF-GEF-dependent endomembrane transport cannot be manipulated with BFA.
Endomembrane system
Brefeldin A
ADP ribosylation factor
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Endomembrane system
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Effects of brefeldin A (BFA) and nordihydroguaiaretic acid (NDGA) on endomembrane structures and lipid synthesis were compared in maize root cells and tobacco Bright Yellow-2 cells. Immunofluorescence and electron microscopy studies showed that NDGA altered the structure and distribution of the endoplasmic reticulum (ER) within 1 h but not of the Golgi apparatus whereas, as shown previously, BFA altered that organization of the Golgi apparatus and, only subsequently, of the ER. Biochemical studies revealed that both drugs and especially BFA led to a strong inhibition of the phytosterol biosynthetic pathway: BFA led to accumulation of sterol precursors. The importance of phytosterols in membrane architecture and membrane trafficking is discussed.
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Nordihydroguaiaretic acid
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Summary Prenylation is necessary for association of the petunia calmodulin CaM53 with the plasma membrane. To determine whether post‐prenylation processing of the protein was also required for plasma membrane targeting, we studied the subcellular localization of a GFP‐labelled CaM53 reporter in yeast and plant cells. Blocking of carboxyl‐methylation of prenylated proteins either by a specific inhibitor or in mutant yeast cells resulted in localization of green fluorescence to what appears to be the endomembrane system, in contrast with the plasma membrane localization observed in control cells. We show that a prenyl‐cysteine methyltransferase (PCM) activity that carboxyl‐methylates prenylated CaM53 also exists in plant cells, and that it is required for efficient plasma membrane targeting. We also report an Arabidopsis gene with homology to PCM and demonstrate that it encodes a protein with PCM activity that localizes to the endomembrane system of plant cells, similar to prenylated but unmethylated CaM53. Together, our data suggest that, following prenylation, CaM53 is probably associated with the endomembrane system, where a PCM activity methylates the prenylated protein prior to targeting it to its final destination in the plasma membrane.
Endomembrane system
Geranylgeranylation
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Prenylation is necessary for association of the petunia calmodulin CaM53 with the plasma membrane. To determine whether post-prenylation processing of the protein was also required for plasma membrane targeting, we studied the subcellular localization of a GFP-labelled CaM53 reporter in yeast and plant cells. Blocking of carboxyl-methylation of prenylated proteins either by a specific inhibitor or in mutant yeast cells resulted in localization of green fluorescence to what appears to be the endomembrane system, in contrast with the plasma membrane localization observed in control cells. We show that a prenyl-cysteine methyltransferase (PCM) activity that carboxyl-methylates prenylated CaM53 also exists in plant cells, and that it is required for efficient plasma membrane targeting. We also report an Arabidopsis gene with homology to PCM and demonstrate that it encodes a protein with PCM activity that localizes to the endomembrane system of plant cells, similar to prenylated but unmethylated CaM53. Together, our data suggest that, following prenylation, CaM53 is probably associated with the endomembrane system, where a PCM activity methylates the prenylated protein prior to targeting it to its final destination in the plasma membrane.
Endomembrane system
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Brefeldin A
Endomembrane system
Daucus carota
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Many proteins are post-translationally modified by lipid moieties such as palmitoyl or prenyl (e.g., farnesyl) groups, creating functional proteolipids. Lipid modifications share the property of increasing a protein's hydrophobicity and thus the propensity of that protein to associate with a membrane. These modifications are used to control the localization and activity of membrane-associated proteins. A well-recognized paradigm is farnesylation of the Ras GTPase that helps target this critical signaling protein to the plasma membrane.
Palmitoylation
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Synaptosomal-associated protein of 25 kDa (SNAP-25) is a palmitoylated membrane protein essential for neurotransmitter release from synaptic terminals. We used neuronal cell lines to study the biosynthesis and posttranslational processing of SNAP-25 to investigate how palmitoylation contributes to the subcellular localization of the protein. SNAP-25 was synthesized as a soluble protein that underwent palmitoylation approximately 20 min after synthesis. Palmitoylation of the protein coincided with its stable membrane association. Treatment of cells with brefeldin A or other disrupters of transport inhibited palmitoylation of newly synthesized SNAP-25 and abolished membrane association. These results demonstrate that the processing of SNAP-25 and its targeting to the plasma membrane depend on an intact transport mechanism along the exocytic pathway. The kinetics of SNAP-25 palmitoylation and membrane association and the sensitivity of these parameters to brefeldin A suggest a novel trafficking pathway for targeting proteins to the plasma membrane. In vitro, SNAP-25 stably associated with membranes was not released from the membrane after chemical deacylation. We propose that palmitoylation of SNAP-25 is required for initial membrane targeting of the protein but that other interactions can maintain membrane association in the absence of fatty acylation.
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Brefeldin A
Transport protein
Gap-43 protein
Peripheral membrane protein
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