scholarly journals Erf4p and Erf2p Form an Endoplasmic Reticulum-associated Complex Involved in the Plasma Membrane Localization of Yeast Ras Proteins

2002 ◽  
Vol 277 (51) ◽  
pp. 49352-49359 ◽  
Author(s):  
Lihong Zhao ◽  
Sandra Lobo ◽  
Xiangwen Dong ◽  
Addison D. Ault ◽  
Robert J. Deschenes
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Membrane Protein ◽  
Secretory Pathway ◽  
Integral Membrane Protein ◽  
Genetic Screen ◽  
Membrane Localization ◽  
Ras Proteins ◽  
Transport Pathway ◽  
Plasma Membrane Localization

Ras oncogene proteins are plasma membrane-associated signal transducers that are found in all eukaryotes. Posttranslational addition of lipid to a carboxyl-terminal CaaXbox (where “C” represents a cysteine, “a” is generally an aliphatic residue, andXcan 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,ERF2andERF4/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.

scholarly journals Palmitoylation and Plasma Membrane Localization of Ras2p by a Nonclassical Trafficking Pathway in Saccharomyces cerevisiae

2003 ◽  
Vol 23 (18) ◽  
pp. 6574-6584 ◽  
Author(s):  
Xiangwen Dong ◽  
David A. Mitchell ◽  
Sandra Lobo ◽  
Lihong Zhao ◽  
Douglas J. Bartels ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Secretory Pathway ◽  
Brefeldin A ◽  
Covalent Attachment ◽  
Membrane Localization ◽  
Ras Proteins ◽  
Plasma Membrane Localization ◽  
Additional Processing ◽  
Endomembrane Trafficking

ABSTRACT 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.

scholarly journals Aberrant substrate engagement of the ER translocon triggers degradation by the Hrd1 ubiquitin ligase

2012 ◽  
Vol 197 (6) ◽  
pp. 761-773 ◽  
Author(s):  
Eric M. Rubenstein ◽  
Stefan G. Kreft ◽  
Wesley Greenblatt ◽  
Robert Swanson ◽  
Mark Hochstrasser
Keyword(s):  
Quality Control ◽  
Endoplasmic Reticulum ◽  
Membrane Protein ◽  
Ubiquitin Ligase ◽  
Apolipoprotein B ◽  
Secretory Pathway ◽  
Protein A ◽  
Proteasomal Degradation ◽  
Membrane Localization ◽  
General Role

Little is known about quality control of proteins that aberrantly or persistently engage the endoplasmic reticulum (ER)-localized translocon en route to membrane localization or the secretory pathway. Hrd1 and Doa10, the primary ubiquitin ligases that function in ER-associated degradation (ERAD) in yeast, target distinct subsets of misfolded or otherwise abnormal proteins based primarily on degradation signal (degron) location. We report the surprising observation that fusing Deg1, a cytoplasmic degron normally recognized by Doa10, to the Sec62 membrane protein rendered the protein a Hrd1 substrate. Hrd1-dependent degradation occurred when Deg1-Sec62 aberrantly engaged the Sec61 translocon channel and underwent topological rearrangement. Mutations that prevent translocon engagement caused a reversion to Doa10-dependent degradation. Similarly, a variant of apolipoprotein B, a protein known to be cotranslocationally targeted for proteasomal degradation, was also a Hrd1 substrate. Hrd1 therefore likely plays a general role in targeting proteins that persistently associate with and potentially obstruct the translocon.

Vid22p, a novel plasma membrane protein, is required for the fructose-1,6-bisphosphatase degradation pathway

2002 ◽  
Vol 115 (3) ◽  
pp. 655-666 ◽  
Author(s):  
C. Randell Brown ◽  
Jameson A. McCann ◽  
Graham Guo-Chiuan Hung ◽  
Christopher P. Elco ◽  
Hui-Ling Chiang
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Secretory Pathway ◽  
Integral Membrane Protein ◽  
Degradation Pathway ◽  
Proteinase K ◽  
Carboxypeptidase Y ◽  
Trafficking Pathway ◽  
Fructose 1,6 Bisphosphatase ◽  
Important Enzyme

Fructose-1,6-bisphosphatase (FBPase), an important enzyme in the gluconeogenic pathway in Saccharomyces cerevisiae, is expressed when cells are grown in media containing a poor carbon source. Following glucose replenishment, FBPase is targeted from the cytosol to intermediate Vid(vacuole import and degradation) vesicles and then to the vacuole for degradation. Recently, several vid mutants that are unable to degrade FBPase in response to glucose were identified. Here, we present VID22, a novel gene involved in FBPase degradation. VID22encodes a glycosylated integral membrane protein that localizes to the plasma membrane. Newly synthesized Vid22p was found in the cytoplasm and then targeted to the plasma membrane independent of the classical secretory pathway. A null mutation of VID22 failed to degrade FBPase following a glucose shift and accumulated FBPase in the cytosol. Furthermore, the majority of FBPase remained in a proteinase K sensitive compartment in the Δvid22 mutant, implying that VID22 is involved in FBPase transport from the cytosol to Vid vesicles. By contrast,starvation-induced autophagy and peroxisome degradation were not impaired in the Δvid22 mutant. This strain also exhibited the proper processing of carboxypeptidase Y and aminopeptidase I in the vacuole. Therefore, Vid22p appears to play a specific role in the FBPase trafficking pathway.

Recent Advances in Developing K-Ras Plasma Membrane Localization Inhibitors

2019 ◽  
Vol 19 (23) ◽  
pp. 2114-2127 ◽  
Author(s):  
Na Ye ◽  
Qingfeng Xu ◽  
Wanwan Li ◽  
Pingyuan Wang ◽  
Jia Zhou
Keyword(s):  
Plasma Membrane ◽  
Cell Growth ◽  
High Throughput Screening ◽  
Drug Repurposing ◽  
Membrane Localization ◽  
Clinical Use ◽  
Ras Proteins ◽  
Plasma Membrane Localization ◽  
Recent Advances ◽  
Specific Inhibitors

: The Ras proteins play an important role in cell growth, differentiation, proliferation and survival by regulating diverse signaling pathways. Oncogenic mutant K-Ras is the most frequently mutated class of Ras superfamily that is highly prevalent in many human cancers. Despite intensive efforts to combat various K-Ras-mutant-driven cancers, no effective K-Ras-specific inhibitors have yet been approved for clinical use to date. Since K-Ras proteins must be associated to the plasma membrane for their function, targeting K-Ras plasma membrane localization represents a logical and potentially tractable therapeutic approach. Here, we summarize the recent advances in the development of K-Ras plasma membrane localization inhibitors including natural product-based inhibitors achieved from high throughput screening, fragment-based drug design, virtual screening, and drug repurposing as well as hit-to-lead optimizations.

scholarly journals PtdIns4P synthesis by PI4KIIIα at the plasma membrane and its impact on plasma membrane identity

2012 ◽  
Vol 199 (6) ◽  
pp. 1003-1016 ◽  
Author(s):  
Fubito Nakatsu ◽  
Jeremy M. Baskin ◽  
Jeeyun Chung ◽  
Lukas B. Tanner ◽  
Guanghou Shui ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Membrane Localization ◽  
Plasma Membrane Localization ◽  
Metabolic Conversion ◽  
Evolutionarily Conserved ◽  
Peripheral Membrane Protein ◽  
Higher Eukaryotes ◽  
Definition Of ◽  
Modest Reduction

Plasma membrane phosphatidylinositol (PI) 4-phosphate (PtdIns4P) has critical functions via both direct interactions and metabolic conversion to PI 4,5-bisphosphate (PtdIns(4,5)P2) and other downstream metabolites. However, mechanisms that control this PtdIns4P pool in cells of higher eukaryotes remain elusive. PI4KIIIα, the enzyme thought to synthesize this PtdIns4P pool, is reported to localize in the ER, contrary to the plasma membrane localization of its yeast homologue, Stt4. In this paper, we show that PI4KIIIα was targeted to the plasma membrane as part of an evolutionarily conserved complex containing Efr3/rolling blackout, which we found was a palmitoylated peripheral membrane protein. PI4KIIIα knockout cells exhibited a profound reduction of plasma membrane PtdIns4P but surprisingly only a modest reduction of PtdIns(4,5)P2 because of robust up-regulation of PtdIns4P 5-kinases. In these cells, however, much of the PtdIns(4,5)P2 was localized intracellularly, rather than at the plasma membrane as in control cells, along with proteins typically restricted to this membrane, revealing a major contribution of PI4KIIIα to the definition of plasma membrane identity.

Cytoplasmic targeting signals mediate delivery of phospholemman to the plasma membrane

2006 ◽  
Vol 290 (5) ◽  
pp. C1275-C1286 ◽  
Author(s):  
Kristan L. Lansbery ◽  
Lauren C. Burcea ◽  
Margaretta L. Mendenhall ◽  
Robert W. Mercer
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Mdck Cells ◽  
Protein Family ◽  
Site Directed Mutagenesis ◽  
Membrane Localization ◽  
Cardiac Sarcolemma ◽  
Plasma Membrane Localization ◽  
The Family ◽  
Corticosteroid Hormone

The FXYD protein family consists of several small, single-span membrane proteins that exhibit a high degree of homology. The best-known members of the family include the γ-subunit of the Na+-K+-ATPase and phospholemman (PLM), a phosphoprotein of cardiac sarcolemma. Other members of the family include corticosteroid hormone-induced factor (CHIF), mammary tumor protein of 8 kDa (Mat-8), and related to ion channels (RIC). The exact physiological roles of the FXYD proteins remain unknown. To better characterize the function of the members of the FXYD protein family, we expressed several members of the family in Madin-Darby canine kidney (MDCK) cells. All of the FXYD proteins, with the exception of PLM, were primarily found in the basolateral plasma membrane. Surprisingly, PLM, a previously characterized plasma membrane protein, was found to colocalize with the endoplasmic reticulum marker protein disulfide isomerase. Treatment of MDCK cells expressing PLM with an agonist of PKC caused some of the PLM to be redistributed to the plasma membrane. Site-directed mutagenesis of residues within the cytoplasmic domain of PLM indicated that a negative charge at Ser69 is necessary to shift the localization of PLM to the plasma membrane. In addition, other regions of PLM necessary for either its endoplasmic reticulum or plasma membrane localization have been elucidated. In contrast to PLM, the plasma membrane localization of CHIF and RIC was not altered by mutation of potential cytoplasmic phosphorylation sites. Overall, these results suggest that phosphorylation of specific residues of PLM may direct PLM from an intracellular compartment to the plasma membrane.

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