scholarly journals Mutational analysis of the intramembranous H10 loop of yeast Nhx1 reveals a critical role in ion homoeostasis and vesicle trafficking

2006 ◽  
Vol 398 (1) ◽  
pp. 97-105 ◽  
Author(s):  
Sanchita Mukherjee ◽  
Laura Kallay ◽  
Christopher L. Brett ◽  
Rajini Rao
Keyword(s):  
Plasma Membrane ◽  
Mutational Analysis ◽  
Vesicle Trafficking ◽  
Critical Role ◽  
Physiological Role ◽  
Carboxypeptidase Y ◽  
Loss Of Function ◽  
Transmembrane Segments ◽  
Tightly Coupled ◽  
Trafficking Defects

Yeast Nhx1 [Na+(K+)/H+ exchanger 1] is an intracellular Na+(K+)/H+ exchanger, localizing to the late endosome where it is important for ion homoeostasis and vesicle trafficking. Phylogenetic analysis of NHE (Na+/H+ exchanger) sequences has ident-ified orthologous proteins, including HsNHE6 (human NHE6), HsNHE7 and HsNHE9 of unknown physiological role. These appear distinct from well-studied mammalian plasma membrane isoforms (NHE1–NHE5). To explore the differences between plasma membrane and intracellular NHEs and understand the link between ion homoeostasis and vesicle trafficking, we examined the consequence of replacing residues in the intramembranous H10 loop of Nhx1 between transmembrane segments 9 and 10. The critical role for the carboxy group of Glu355 in ion transport is consistent with the invariance of this residue in all NHEs. Surprisingly, residues specifically conserved in the intracellular isoforms (such as Phe357 and Tyr361) could not be replaced with closely similar residues (leucine and phenylalanine) found in the plasma membrane isoforms without loss of function, revealing unexpected side chain specificity. The trafficking phenotypes of all Nhx1 mutants, including hygromycin-sensitivity and missorting of carboxypeptidase Y, were found to directly correlate with pH homoeostasis defects and could be proportionately corrected by titration with weak base. The present study demonstrates the importance of the H10 loop of the NHE family, highlights the differences between plasma membrane and intracellular isoforms and shows that trafficking defects are tightly coupled with pH homoeostasis.

scholarly journals MRTF transcription and Ezrin-dependent plasma membrane blebbing are required for entotic invasion

2017 ◽  
Vol 216 (10) ◽  
pp. 3087-3095 ◽  
Author(s):  
Laura Soto Hinojosa ◽  
Manuel Holst ◽  
Christian Baarlink ◽  
Robert Grosse
Keyword(s):  
Plasma Membrane ◽  
Cell Invasion ◽  
Serum Response Factor ◽  
Critical Role ◽  
Actin Dynamics ◽  
Actin Cortex ◽  
Tightly Coupled ◽  
Related Transcription Factor ◽  
Nuclear Shuttling ◽  
The Impact

Entosis is a nonapoptotic form of cell death initiated by actomyosin-dependent homotypic cell-in-cell invasion that can be observed in malignant exudates during tumor progression. We previously demonstrated formin-mediated actin dynamics at the rear of the invading cell as well as nonapoptotic plasma membrane (PM) blebbing in this cellular motile process. Although the contractile actin cortex involved in bleb-driven motility is well characterized, a role for transcriptional regulation in this process has not been studied. Here, we explore the impact of the actin-controlled MRTF–SRF (myocardin-related transcription factor–serum response factor) pathway for sustained PM blebbing and entotic invasion. We find that cortical blebbing is tightly coupled to MRTF nuclear shuttling to promote the SRF transcriptional activity required for entosis. Furthermore, PM blebbing triggered SRF-mediated up-regulation of the metastasis-associated ERM protein Ezrin. Notably, Ezrin is sufficient and important to sustain bleb dynamics for cell-in-cell invasion when SRF is suppressed. Our results highlight the critical role of the actin-regulated MRTF transcriptional pathway for bleb-associated invasive motility, such as during entosis.

scholarly journals Topology of transmembrane segments 1–4 in the human chloride/bicarbonate anion exchanger 1 (AE1) by scanning N-glycosylation mutagenesis

2005 ◽  
Vol 390 (1) ◽  
pp. 137-144 ◽  
Author(s):  
Joanne C. Cheung ◽  
Jing Li ◽  
Reinhart A. F. Reithmeier
Keyword(s):  
Plasma Membrane ◽  
Anion Exchanger ◽  
Physiological Role ◽  
Mutant Form ◽  
Extracellular Loop ◽  
Anion Exchanger 1 ◽  
Hek 293 ◽  
Human Embryonic Kidney Cells ◽  
Transmembrane Segments

Human AE1 (anion exchanger 1), or Band 3, is an abundant membrane glycoprotein found in the plasma membrane of erythrocytes. The physiological role of the protein is to carry out chloride/bicarbonate exchange across the plasma membrane, a process that increases the carbon-dioxide-carrying capacity of blood. To study the topology of TMs (transmembrane segments) 1–4, a series of scanning N-glycosylation mutants were created spanning the region from EC (extracellular loop) 1 to EC2 in full-length AE1. These constructs were expressed in HEK-293 (human embryonic kidney) cells, and their N-glycosylation efficiencies were determined. Unexpectedly, positions within putative TMs 2 and 3 could be efficiently glycosylated. In contrast, the same positions were very poorly glycosylated when present in mutant AE1 with the SAO (Southeast Asian ovalocytosis) deletion (ΔA400–A408) in TM1. These results suggest that the TM2–3 region of AE1 may become transiently exposed to the endoplasmic reticulum lumen during biosynthesis, and that there is a competition between proper folding of the region into the membrane and N-glycosylation at introduced sites. The SAO deletion disrupts the proper integration of TMs 1–2, probably leaving the region exposed to the cytosol. As a result, engineered N-glycosylation acceptor sites in TM2–3 could not be utilized by the oligosaccharyltransferase in this mutant form of AE1. The properties of TM2–3 suggest that these segments form a re-entrant loop in human AE1.

scholarly journals Membrane chaperone Shr3 assists in folding amino acid permeases preventing precocious ERAD

2007 ◽  
Vol 176 (5) ◽  
pp. 617-628 ◽  
Author(s):  
Jhansi Kota ◽  
C. Fredrik Gilstring ◽  
Per O. Ljungdahl
Keyword(s):  
Amino Acid ◽  
Critical Role ◽  
Amino Acid Uptake ◽  
Functional Expression ◽  
Coupled Processes ◽  
Acid Uptake ◽  
Transmembrane Segments ◽  
Large Molecular Weight ◽  
Tightly Coupled ◽  
Amino Acid Permeases

The yeast endoplasmic reticulum (ER) membrane-localized chaperone Shr3 plays a critical role in enabling amino acid permeases (AAPs) to fold and attain proper structures required for functional expression at the plasma membrane. In the absence of Shr3, AAPs specifically accumulate in the ER, where despite the correct insertion of their 12 transmembrane segments (TMSs), they aggregate forming large molecular weight complexes. We show that Shr3 prevents aggregation and facilitates the functional assembly of independently coexpressed N- and C-terminal fragments of the general AAP Gap1. Shr3 interacts with and maintains the first five TMSs in a conformation that can posttranslationally assemble with the remaining seven TMSs. We also show that Doa10- and Hrd1-dependent ER-associated degradation (ERAD) pathways redundantly degrade AAP aggregates. In combination, doa10Δ hrd1Δ mutations stabilize AAP aggregates and partially suppress amino acid uptake defects of shr3 mutants. Consequently, in cells with impaired ERAD, AAPs are able to attain functional conformations independent of Shr3. These findings illustrate that folding and degradation are tightly coupled processes during membrane protein biogenesis.

Proline residues in two tightly coupled helices of the sulphate transporter, SHST1, are important for sulphate transport

2001 ◽  
Vol 356 (2) ◽  
pp. 589-594 ◽  
Author(s):  
Megan C. SHELDEN ◽  
Patrick LOUGHLIN ◽  
M. Louise TIERNEY ◽  
Susan M. HOWITT
Keyword(s):  
Plasma Membrane ◽  
Critical Role ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Transmembrane Helices ◽  
Transport Activity ◽  
Post Translational Modification ◽  
Sulphate Transport ◽  
Stylosanthes Hamata ◽  
Tightly Coupled

The sulphate transporter SHST1, from Stylosanthes hamata, features three tightly coupled transmembrane helices which include proline residues that are conserved in most related transporters. We used site-directed mutagenesis and expression of the mutant transporters in yeast to test whether these proline residues are important for function. Four proline residues were replaced by both alanine and leucine. Only one of these proline residues, Pro-144, was essential for sulphate transport. However, mutation of either Pro-133 or Pro-160 resulted in a severe decrease in sulphate transport activity; this was due more to a decrease in transport activity than to a decrease in the amount of mutant SHST1 in the plasma membrane. These results suggest that all three proline residues are important for transport, and that the conformation of the three tightly coupled helices may play a critical role in sulphate transport. We also show that SHST1 undergoes a post-translational modification that is required for trafficking to the plasma membrane.

scholarly journals TAL effectors with avirulence activity in African strains of Xanthomonas oryzae pv. oryzae

2021 ◽  
Author(s):  
Marlene Lachaux ◽  
Emilie Thomas ◽  
Adam J Bogdanove ◽  
Boris Szurek ◽  
Mathilde Hutin
Keyword(s):  
Mutational Analysis ◽  
Critical Role ◽  
Xanthomonas Oryzae ◽  
Disease Development ◽  
Loss Of Function ◽  
Transcription Activator ◽  
Activation Domain ◽  
Rice Varieties ◽  
Resistance Response ◽  
Asian Strain

Background: Xanthomonas oryzae pv. oryzae causes bacterial leaf blight, a devastating disease of rice. Among the type-3 effectors secreted by Xanthomonas oryzae pv. oryzae to support pathogen virulence, the Transcription Activator-Like Effector (TALE) family plays a critical role. Some TALEs are major virulence factors that activate susceptibility (S) genes, overexpression of which contributes to disease development. Host incompatibility can result from TALE-induced expression of so-called executor (E) genes leading to a strong and rapid resistance response that blocks disease development. In that context, the TALE functions as an avirulence (Avr) factor. To date no such avirulence factors have been identified in African strains of Xanthomonas oryzae pv. oryzae. Results: With respect to the importance of TALEs in the Rice-Xoo pathosystem, we aimed at identifying those that may act as Avr factor within African Xoo. We screened 86 rice accessions, and identified 12 that were resistant to two African strains while being susceptible to a well-studied Asian strain. In a gain of function approach based on the introduction of each of the nine tal genes of the avirulent African strain MAI1 into the virulent Asian strain PXO99A, four were found to trigger resistance on specific rice accessions. Loss-of-function mutational analysis further demonstrated the avr activity of two of them, talD and talI, on the rice varieties IR64 and CT13432 respectively. Further analysis of TalI demonstrated the requirement of its activation domain for triggering resistance in CT13432. Resistance in 9 of the 12 rice accessions that were resistant against African Xoo specifically, including CT13432, could be suppressed or largely suppressed by trans-expression of the truncTALE tal2h, similarly to resistance conferred by the Xa1 gene which recognizes TALEs generally independently of their activation domain. Conclusion: We identified and characterized TalD and TalI as two African Xoo TALEs with avirulence activity on IR64 and CT13432 respectively. Resistance of CT13432 against African Xoo results from the combination of two mechanisms, one relying on the TalI-mediated induction of an unknown executor gene and the other on an Xa1-like gene or allele.

scholarly journals Topological and Mutational Analysis ofSaccharomyces cerevisiaeSte14p, Founding Member of the Isoprenylcysteine Carboxyl Methyltransferase Family

2001 ◽  
Vol 12 (7) ◽  
pp. 1957-1971 ◽  
Author(s):  
Julia D. Romano ◽  
Susan Michaelis
Keyword(s):  
Mutational Analysis ◽  
Critical Role ◽  
Random Mutagenesis ◽  
Open Reading Frames ◽  
Site Directed Mutagenesis ◽  
Endoplasmic Reticulum Membrane ◽  
Loss Of Function ◽  
Founding Member ◽  
Carboxyl Methyltransferase ◽  
Isoprenylcysteine Carboxyl Methyltransferase

Eukaryotic proteins that terminate in a CaaX motif undergo three processing events: isoprenylation, C-terminal proteolytic cleavage, and carboxyl methylation. In Saccharomyces cerevisiae, the latter step is mediated by Ste14p, an integral endoplasmic reticulum membrane protein. Ste14p is the founding member of the isoprenylcysteine carboxyl methyltransferase (ICMT) family, whose members share significant sequence homology. Because the physiological substrates of Ste14p, such as Ras and the yeast a-factor precursor, are isoprenylated and reside on the cytosolic side of membranes, the Ste14p residues involved in enzymatic activity are predicted to be cytosolically disposed. In this study, we have investigated the topology of Ste14p by analyzing the protease protection of epitope-tagged versions of Ste14p and the glycosylation status of Ste14p-Suc2p fusions. Our data lead to a topology model in which Ste14p contains six membrane spans, two of which form a helical hairpin. According to this model most of the Ste14p hydrophilic regions are located in the cytosol. We have also generated ste14mutants by random and site-directed mutagenesis to identify residues of Ste14p that are important for activity. Notably, four of the five loss-of-function mutations arising from random mutagenesis alter residues that are highly conserved among the ICMT family. Finally, we have identified a novel tripartite consensus motif in the C-terminal region of Ste14p. This region is similar among all ICMT family members, two phospholipid methyltransferases, several ergosterol biosynthetic enzymes, and a group of bacterial open reading frames of unknown function. Site-directed and random mutations demonstrate that residues in this region play a critical role in the function of Ste14p.

scholarly journals RNF141 interacts with KRAS to promote colorectal cancer progression

Oncogene ◽  
2021 ◽  
Author(s):  
Jiuna Zhang ◽  
Xiaoyu Jiang ◽  
Jie Yin ◽  
Shiying Dou ◽  
Xiaoli Xie ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Plasma Membrane ◽  
Tumor Growth ◽  
Cancer Progression ◽  
Critical Role ◽  
Ring Finger ◽  
Immunohistochemical Analysis ◽  
Bimolecular Fluorescence Complementation

AbstractRING finger proteins (RNFs) play a critical role in cancer initiation and progression. RNF141 is a member of RNFs family; however, its clinical significance, roles, and mechanism in colorectal cancer (CRC) remain poorly understood. Here, we examined the expression of RNF141 in 64 pairs of CRC and adjacent normal tissues by real-time PCR, Western blot, and immunohistochemical analysis. We found that there was more expression of RNF141 in CRC tissue compared with its adjacent normal tissue and high RNF141 expression associated with T stage. In vivo and in vitro functional experiments were conducted and revealed the oncogenic role of RNF141 in CRC. RNF141 knockdown suppressed proliferation, arrested the cell cycle in the G1 phase, inhibited migration, invasion and HUVEC tube formation but promoted apoptosis, whereas RNF141 overexpression exerted the opposite effects in CRC cells. The subcutaneous xenograft models showed that RNF141 knockdown reduced tumor growth, but its overexpression promoted tumor growth. Mechanistically, liquid chromatography-tandem mass spectrometry indicated RNF141 interacted with KRAS, which was confirmed by Co-immunoprecipitation, Immunofluorescence assay. Further analysis with bimolecular fluorescence complementation (BiFC) and Glutathione-S-transferase (GST) pull-down assays showed that RNF141 could directly bind to KRAS. Importantly, the upregulation of RNF141 increased GTP-bound KRAS, but its knockdown resulted in a reduction accordingly. Next, we demonstrated that RNF141 induced KRAS activation via increasing its enrichment on the plasma membrane not altering total KRAS expression, which was facilitated by the interaction with LYPLA1. Moreover, KRAS silencing partially abolished the effect of RNF141 on cell proliferation and apoptosis. In addition, our findings presented that RNF141 functioned as an oncogene by upregulating KRAS activity in a manner of promoting KRAS enrichment on the plasma membrane in CRC.

scholarly journals Parkin interacting substrate zinc finger protein 746 is a pathological mediator in Parkinson’s disease

Brain ◽  
2019 ◽  
Vol 142 (8) ◽  
pp. 2380-2401 ◽  
Author(s):  
Saurav Brahmachari ◽  
Saebom Lee ◽  
Sangjune Kim ◽  
Changqing Yuan ◽  
Senthilkumar S Karuppagounder ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Zinc Finger ◽  
Zinc Finger Protein ◽  
Critical Role ◽  
Trna Synthetase ◽  
Loss Of Function ◽  
Substrate Protein ◽  
Finger Protein ◽  
Sporadic Parkinson’S Disease

Abstract α-Synuclein misfolding and aggregation plays a major role in the pathogenesis of Parkinson’s disease. Although loss of function mutations in the ubiquitin ligase, parkin, cause autosomal recessive Parkinson’s disease, there is evidence that parkin is inactivated in sporadic Parkinson’s disease. Whether parkin inactivation is a driver of neurodegeneration in sporadic Parkinson’s disease or a mere spectator is unknown. Here we show that parkin in inactivated through c-Abelson kinase phosphorylation of parkin in three α-synuclein-induced models of neurodegeneration. This results in the accumulation of parkin interacting substrate protein (zinc finger protein 746) and aminoacyl tRNA synthetase complex interacting multifunctional protein 2 with increased parkin interacting substrate protein levels playing a critical role in α-synuclein-induced neurodegeneration, since knockout of parkin interacting substrate protein attenuates the degenerative process. Thus, accumulation of parkin interacting substrate protein links parkin inactivation and α-synuclein in a common pathogenic neurodegenerative pathway relevant to both sporadic and familial forms Parkinson’s disease. Thus, suppression of parkin interacting substrate protein could be a potential therapeutic strategy to halt the progression of Parkinson’s disease and related α-synucleinopathies.

scholarly journals Functional Role of Charged Residues in the Transmembrane Segments of the Yeast Plasma Membrane H+-ATPase

2000 ◽  
Vol 275 (21) ◽  
pp. 15709-15716 ◽  
Author(s):  
Valery V. Petrov ◽  
Kristine P. Padmanabha ◽  
Robert K. Nakamoto ◽  
Kenneth E. Allen ◽  
Carolyn W. Slayman
Keyword(s):  
Plasma Membrane ◽  
Functional Role ◽  
Yeast Plasma Membrane ◽  
Transmembrane Segments ◽  
Charged Residues

scholarly journals Proprotein Convertase 5/6 Is Critical for Embryo Implantation in Women: Regulating Receptivity by Cleaving EBP50, Modulating Ezrin Binding, and Membrane-Cytoskeletal Interactions

Endocrinology ◽  
2011 ◽  
Vol 152 (12) ◽  
pp. 5041-5052 ◽  
Author(s):  
Sophea Heng ◽  
Ana Cervero ◽  
Carlos Simon ◽  
Andrew N. Stephens ◽  
Ying Li ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cellular Localization ◽  
Structural Changes ◽  
Current Knowledge ◽  
Embryo Implantation ◽  
Critical Role ◽  
Human Endometrium ◽  
Scaffolding Protein ◽  
Proprotein Convertase ◽  
Proteomic Approach

Establishment of endometrial receptivity is vital for successful embryo implantation; its failure causes infertility. Epithelial receptivity acquisition involves dramatic structural changes in the plasma membrane and cytoskeleton. Proprotein convertase 5/6 (PC6), a serine protease of the proprotein convertase (PC) family, is up-regulated in the human endometrium specifically at the time of epithelial receptivity and stromal cell decidualization. PC6 is the only PC member tightly regulated in this manner. The current study addressed the importance and mechanisms of PC6 action in regulating receptivity in women. PC6 was dysregulated in the endometrial epithelium during the window of implantation in infertile women of three demographically different cohorts. Its critical role in receptivity was evidenced by a significant reduction in mouse blastocyst attachment of endometrial epithelial cells after PC6 knockdown by small interfering RNA. Using a proteomic approach, we discovered that PC6 cleaved the key scaffolding protein, ezrin-radixin-moesin binding phosphoprotein 50 (EBP50), thereby profoundly affecting its interaction with binding protein ezrin (a key protein bridging actin filaments and plasma membrane), EBP50/ezrin cellular localization, and cytoskeleton-membrane connections. We further validated this novel PC6 regulation of receptivity in human endometrium in vivo in fertile vs. infertile patients. These results strongly indicate that PC6 plays a key role in regulating fundamental cellular remodeling processes, such as plasma membrane transformation and membrane-cytoskeletal interface reorganization. PC6 cleavage of a crucial scaffolding protein EBP50, thereby profoundly regulating membrane-cytoskeletal reorganization, greatly extends the current knowledge of PC biology and provides substantial new mechanistic insight into the fields of reproduction, basic cellular biology, and PC biochemistry.

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