scholarly journals Membrane insertion of gap junction connexins: polytopic channel forming membrane proteins.

1994 ◽  
Vol 127 (2) ◽  
pp. 343-355 ◽  
Author(s):  
M M Falk ◽  
N M Kumar ◽  
N B Gilula
Keyword(s):  
Plasma Membrane ◽  
Gap Junction ◽  
Proteolytic Processing ◽  
Signal Peptidase ◽  
Membrane Insertion ◽  
Competent Cell ◽  
In Vitro Synthesis ◽  
Amino Terminal

Connexins, the proteins that form gap junction channels, are polytopic plasma membrane (PM) proteins that traverse the plasma membrane bilayer four times. The insertion of five different connexins into the membrane of the ER was studied by synthesizing connexins in translation-competent cell lysates supplemented with pancreatic ER-derived microsomes, and by expressing connexins in vivo in several eucaryotic cell types. In addition, the subcellular distribution of the connexins was determined. In vitro-synthesis in the presence of microsomes resulted in the signal recognition particle-dependent membrane insertion of the connexins. The membrane insertion of all connexins was accompanied by an efficient proteolytic processing that was dependent on the microsome concentration. Endogenous unprocessed connexins were detectable in the microsomes used, indicating that the pancreatic microsomes serve as a competent recipient in vivo for unprocessed full length connexins. Although oriented with their amino terminus in the cytoplasm, the analysis of the cleavage reaction indicated that an unprecedented processing by signal peptidase resulted in the removal of an amino-terminal portion of the connexins. Variable amounts of similar connexin cleavage products were also identified in the ER membranes of connexin overexpressing cells. The amount generated correlated with the level of protein expression. These results demonstrate that the connexins contain a cryptic signal peptidase cleavage site that can be processed by this enzyme in vitro and in vivo in association with their membrane insertion. Consequently, a specific factor or condition must be required to prevent this aberrant processing of connexins under normal conditions in the cell.

scholarly journals Mechanisms of plasma membrane targeting of formin mDia2 through its amino terminal domains

2011 ◽  
Vol 22 (2) ◽  
pp. 189-201 ◽  
Author(s):  
Roman Gorelik ◽  
Changsong Yang ◽  
Vasumathi Kameswaran ◽  
Roberto Dominguez ◽  
Tatyana Svitkina
Keyword(s):  
Plasma Membrane ◽  
Electrostatic Interactions ◽  
Coiled Coil ◽  
Small Gtpases ◽  
Coincidence Detection ◽  
Membrane Targeting ◽  
Amino Terminal ◽  
N Terminus

The formin mDia2 mediates the formation of lamellipodia and filopodia during cell locomotion. The subcellular localization of activated mDia2 depends on interactions with actin filaments and the plasma membrane. We investigated the poorly understood mechanism of plasma membrane targeting of mDia2 and found that the entire N-terminal region of mDia2 preceding the actin-polymerizing formin homology domains 1 and 2 (FH1–FH2) module was potently targeted to the membrane. This localization was enhanced by Rif, but not by other tested small GTPases, and depended on a positively charged N-terminal basic domain (BD). The BD bound acidic phospholipids in vitro, suggesting that in vivo it may associate with the plasma membrane through electrostatic interactions. Unexpectedly, a fragment consisting of the GTPase-binding region and the diaphanous inhibitory domain (G-DID), thought to mediate the interaction with GTPases, was not targeted to the plasma membrane even in the presence of constitutively active Rif. Addition of the BD or dimerization/coiled coil domains to G-DID rescued plasma membrane targeting in cells. Direct binding of Rif to mDia2 N terminus required the presence of both G and DID. These results suggest that the entire N terminus of mDia2 serves as a coincidence detection module, directing mDia2 to the plasma membrane through interactions with phospholipids and activated Rif.

Stimulation of AQP2 membrane insertion in renal epithelial cells in vitro and in vivo by the cGMP phosphodiesterase inhibitor sildenafil citrate (Viagra)

2005 ◽  
Vol 288 (6) ◽  
pp. F1103-F1112 ◽  
Author(s):  
Richard Bouley ◽  
Nuria Pastor-Soler ◽  
Ori Cohen ◽  
Margaret McLaughlin ◽  
Sylvie Breton ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Collecting Duct ◽  
Sildenafil Citrate ◽  
Membrane Insertion ◽  
Vasopressin Receptor ◽  
Pde5 Inhibitors ◽  
Principal Cells ◽  
Medullary Collecting Duct

Vasopressin-stimulated insertion of the aquaporin 2 (AQP2) water channel into the plasma membrane of kidney collecting duct principal cells is a key event in the urinary concentrating mechanism. The paradigm for vasopressin-receptor signaling involves cAMP-mediated protein kinase A activation, which results in the functionally critical phosphorylation of AQP2 on amino acid serine 256. We previously showed that a parallel cGMP-mediated signaling pathway also leads to AQP2 membrane insertion in AQP2-transfected LLC-PK1 (LLC-AQP2) cells and in outer medullary collecting duct principal cells in situ (Bouley R, Breton S, Sun T, McLaughlin M, Nsumu NN, Lin HY, Ausiello DA, and Brown D. J Clin Invest 106: 1115–1126, 2000). In the present report, we show by immunofluorescence microscopy, and Western blotting of plasma membrane fractions, that 45-min exposure of LLC-AQP2 cells to the cGMP phosphodiesterase type 5 (PDE5) inhibitors sildenafil citrate (Viagra) or 4-{[3',4'-methylene-dioxybenzyl]amino}-6-methoxyquinazoline elevates intracellular cGMP levels and results in the plasma membrane accumulation of AQP2; i.e., they mimic the vasopressin effect. Importantly, our data also show that acute exposure to PDE5 inhibitors for 60 min induces apical accumulation of AQP2 in kidney medullary collecting duct principal cells both in tissue slices incubated in vitro as well as in vivo after intravenous injection of Viagra into rats. These data suggest that AQP2 membrane insertion can be induced independently of vasopressin-receptor activation by activating a parallel cGMP-mediated signal transduction pathway with cGMP PDE inhibitors. These results provide proof-of-principle that pharmacological activation of vasopressin-independent, cGMP signaling pathways could aid in the treatment of those forms of nephrogenic diabetes insipidus that are due to vasopressin-2 receptor dysfunction.

scholarly journals In vitro synthesis and membrane insertion of bovine MP26, an integral protein from lens fiber plasma membrane.

1983 ◽  
Vol 96 (3) ◽  
pp. 633-638 ◽  
Author(s):  
D L Paul ◽  
D A Goodenough
Keyword(s):  
Plasma Membranes ◽  
Proteolytic Processing ◽  
Membrane Insertion ◽  
Membrane Association ◽  
Lens Fiber ◽  
In Vitro Synthesis ◽  
Reticulocyte Lysate ◽  
Immune Precipitation ◽  
Dog Pancreas

Synthesis of MP26, the principal protein of lens fiber plasma membranes, was directed in the reticulocyte lysate system by poly A mRNA enriched from whole bovine lens RNA using oligo (dt)-cellulose chromatography. Synthesized MP26 was enriched by immune precipitation. The in vitro-synthesized MP26 had an electrophoretic mobility indistinguishable from that of the native molecule. MP26 showed a cotranslational requirement for dog pancreas microsomes in order for membrane association to occur. Microsome-associated in vitro-synthesized MP26 showed a sensitivity to digestion with chymotrypsin which was similar to the sensitivity of native MP26 in isolated lens fiber plasma membranes, indicating correct insertion of the MP26 into the microsome. Synthesis and membrane insertion of MP26 using N-formyl-[35S]methionyl tRNA as label demonstrated that no proteolytic processing or significant glycosylation accompanied membrane insertion. Chymotryptic cleavage of membrane-inserted, N-formyl-[35S]methionine-labeled MP26 resulted in loss of label, suggesting that the N-terminal of the in vitro-synthesized MP26 faces the cytoplasm.

scholarly journals Molecular Analysis of Phr Peptide Processing in Bacillus subtilis

2003 ◽  
Vol 185 (16) ◽  
pp. 4861-4871 ◽  
Author(s):  
Sophie Stephenson ◽  
Christian Mueller ◽  
Min Jiang ◽  
Marta Perego
Keyword(s):  
Bacillus Subtilis ◽  
Response Regulator ◽  
Signal Peptidase ◽  
Type I ◽  
Peptidase Activity ◽  
Amino Terminal ◽  
Processing Event ◽  
Signal Peptidases

ABSTRACT In Bacillus subtilis, an export-import pathway regulates production of the Phr pentapeptide inhibitors of Rap proteins. Processing of the Phr precursor proteins into the active pentapeptide form is a key event in the initiation of sporulation and competence development. The PhrA (ARNQT) and PhrE (SRNVT) peptides inhibit the RapA and RapE phosphatases, respectively, whose activity is directed toward the Spo0F∼P intermediate response regulator of the sporulation phosphorelay. The PhrC (ERGMT) peptide inhibits the RapC protein acting on the ComA response regulator for competence with regard to DNA transformation. The structural organization of PhrA, PhrE, and PhrC suggested a role for type I signal peptidases in the processing of the Phr preinhibitor, encoded by the phr genes, into the proinhibitor form. The proinhibitor was then postulated to be cleaved to the active pentapeptide inhibitor by an additional enzyme. In this report, we provide evidence that Phr preinhibitor proteins are subject to only one processing event at the peptide bond on the amino-terminal end of the pentapeptide. This processing event is most likely independent of type I signal peptidase activity. In vivo and in vitro analyses indicate that none of the five signal peptidases of B. subtilis (SipS, SipT, SipU, SipV, and SipW) are indispensable for Phr processing. However, we show that SipV and SipT have a previously undescribed role in sporulation, competence, and cell growth.

scholarly journals Membrane insertion of uracil permease, a polytopic yeast plasma membrane protein.

1991 ◽  
Vol 11 (2) ◽  
pp. 1114-1124 ◽  
Author(s):  
S Silve ◽  
C Volland ◽  
C Garnier ◽  
R Jund ◽  
M R Chevallier ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Terminal Segment ◽  
Proteolytic Processing ◽  
Endoplasmic Reticulum Membrane ◽  
Transmembrane Segments ◽  
C Terminus ◽  
Signal Anchor ◽  
Anchor Sequence

Uracil permease is a multispanning protein of the Saccharomyces cerevisiae plasma membrane which is encoded by the FUR4 gene and produced in limited amounts. It has a long N-terminal hydrophilic segment, which is followed by 10 to 12 putative transmembrane segments, and a hydrophilic C terminus. The protein carries seven potential N-linked glycosylation sites, three of which are in its N-terminal segment. Overexpression of this permease and specific antibodies were used to show that uracil permease undergoes neither N-linked glycosylation nor proteolytic processing. Uracil permease N-terminal segments of increasing lengths were fused to a reporter glycoprotein, acid phosphatase. The in vitro and in vivo fates of the resulting hybrid proteins were analyzed to identify the first signal anchor sequence of the permease and demonstrate the cytosolic orientation of its N-terminal hydrophilic sequence. In vivo insertion of the hybrid protein bearing the first signal anchor sequence of uracil permease into the endoplasmic reticulum membrane was severely blocked in sec61 and sec62 translocation mutants.

scholarly journals Fracture healing is delayed in the absence of gasdermin signaling

2021 ◽  
Author(s):  
Gabriel Mbalaviele ◽  
Kai Sun ◽  
Chun Wang ◽  
Jianqiu Xiao ◽  
Michael D Brodt ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fracture Healing ◽  
Inflammatory Responses ◽  
Biomechanical Properties ◽  
Amino Terminal ◽  
Membrane Pores ◽  
Bone Injury ◽  
Bone Callus

Amino-terminal fragments from proteolytically cleaved gasdermins (GSDMs) form plasma membrane pores that enable the secretion of interleukin-1β (IL-1β) and IL-18. Excessive GSDM-mediated pore formation can compromise the integrity of the plasma membrane thereby causing the lytic inflammatory cell death, pyroptosis. We found that GSDMD and GSDME were the only GSDMs that were readily expressed in bone microenvironment. Therefore, we tested the hypothesis that GSDMD and GSDME are implicated in fracture healing owing to their role in the obligatory inflammatory response following injury. We found that bone callus volume and biomechanical properties of injured bones were significantly reduced in mice lacking either GSDM compared with wild-type (WT) mice, indicating that fracture healing was compromised in mutant mice. However, compound loss of GSDMD and GSDME did not exacerbate the outcomes, suggesting shared actions of both GSDMs in fracture healing. Mechanistically, bone injury induced IL-1β and IL-18 secretion in vivo, a response that was mimicked in vitro by bone debris and ATP, which function as inflammatory danger signals. Importantly, the secretion of these cytokines was attenuated in conditions of GSDMD deficiency. Finally, deletion of IL-1 receptor reproduced the phenotype of Gsdmd or Gsdme deficient mice, implying that inflammatory responses induced by the GSDM-IL-1 axis promote bone healing after fracture.

Membrane insertion of uracil permease, a polytopic yeast plasma membrane protein

1991 ◽  
Vol 11 (2) ◽  
pp. 1114-1124
Author(s):  
S Silve ◽  
C Volland ◽  
C Garnier ◽  
R Jund ◽  
M R Chevallier ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Terminal Segment ◽  
Proteolytic Processing ◽  
Endoplasmic Reticulum Membrane ◽  
Transmembrane Segments ◽  
C Terminus ◽  
Signal Anchor ◽  
Anchor Sequence

Uracil permease is a multispanning protein of the Saccharomyces cerevisiae plasma membrane which is encoded by the FUR4 gene and produced in limited amounts. It has a long N-terminal hydrophilic segment, which is followed by 10 to 12 putative transmembrane segments, and a hydrophilic C terminus. The protein carries seven potential N-linked glycosylation sites, three of which are in its N-terminal segment. Overexpression of this permease and specific antibodies were used to show that uracil permease undergoes neither N-linked glycosylation nor proteolytic processing. Uracil permease N-terminal segments of increasing lengths were fused to a reporter glycoprotein, acid phosphatase. The in vitro and in vivo fates of the resulting hybrid proteins were analyzed to identify the first signal anchor sequence of the permease and demonstrate the cytosolic orientation of its N-terminal hydrophilic sequence. In vivo insertion of the hybrid protein bearing the first signal anchor sequence of uracil permease into the endoplasmic reticulum membrane was severely blocked in sec61 and sec62 translocation mutants.

scholarly journals Novel Proteolytic Processing of the Ectodomain of the Zinc Transporter ZIP4 (SLC39A4) during Zinc Deficiency Is Inhibited by Acrodermatitis Enteropathica Mutations

2008 ◽  
Vol 29 (1) ◽  
pp. 129-139 ◽  
Author(s):  
Taiho Kambe ◽  
Glen K. Andrews
Keyword(s):  
Plasma Membrane ◽  
Zinc Deficiency ◽  
Zinc Transporter ◽  
Proteolytic Processing ◽  
Hek293 Cells ◽  
Acrodermatitis Enteropathica ◽  
Amino Terminal ◽  
Caco2 Cells ◽  
Carboxyl Terminal

ABSTRACT The zinc transporter ZIP4 (SLC39A4) is mutated in humans with the rare, autosomal recessive genetic disease acrodermatitis enteropathica. In mice, this gene is essential during early embryonic development. ZIP4 is dynamically regulated by multiple posttranscriptional mechanisms, and studies of mouse ZIP4 reported herein reveal that the ectodomain, the extracellular amino-terminal half of the protein, is proteolytically removed during prolonged zinc deficiency while the remaining eight-transmembrane carboxyl-terminal half of the protein is accumulated on the plasma membrane as an abundant form of ZIP4. This novel ZIP4 processing occurs in vivo in the intestine and visceral endoderm, in mouse Hepa cells that express the endogenous Slc39a4 gene and in transfected MDCK and CaCo2 cells, but not HEK293 cells. In transfected MDCK and CaCo2 cells, the ectodomain accumulated and remained associated with membranes when zinc was deficient. ZIP4 cleavage was attenuated by inhibitors of endocytosis, which suggests that the processed protein is recycled back to the plasma membrane and that the ectodomain may be internalized. Ectodomain cleavage is inhibited by acrodermatitis enteropathica mutations near a predicted metalloproteinase cleavage site which is also essential for proper ectodomain cleavage, and overexpression of processed ZIP4 or ZIP4 with ectodomain truncations rendered the mouse Mt1 gene hypersensitive to zinc. These finding suggest that the processing of ZIP4 may represent a significant regulatory mechanism controlling its function.

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.

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