Postprenylation CAAX Processing Is Required for Proper Localization of Ras but Not Rho GTPases

The CAAX motif at the C terminus of most monomeric GTPases is required for membrane targeting because it signals for a series of three posttranslational modifications that include isoprenylation, endoproteolytic release of the C-terminal– AAX amino acids, and carboxyl methylation of the newly exposed isoprenylcysteine. The individual contributions of these modifications to protein trafficking and function are unknown. To address this issue, we performed a series of experiments with mouse embryonic fibroblasts (MEFs) lacking Rce1 (responsible for removal of the –AAX sequence) or Icmt (responsible for carboxyl methylation of the isoprenylcysteine). In MEFs lacking Rce1 or Icmt, farnesylated Ras proteins were mislocalized. In contrast, the intracellular localizations of geranylgeranylated Rho GTPases were not perturbed. Consistent with the latter finding, RhoGDI binding and actin remodeling were normal in Rce1- and Icmt-deficient cells. Swapping geranylgeranylation for farnesylation on Ras proteins or vice versa on Rho proteins reversed the differential sensitivities to Rce1 and Icmt deficiency. These results suggest that postprenylation CAAX processing is required for proper localization of farnesylated Ras but not geranygeranylated Rho proteins.

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Posttranslational Modifications and the Immunogenicity of Biotherapeutics

Journal of Immunology Research ◽

10.1155/2016/5358272 ◽

2016 ◽

Vol 2016 ◽

pp. 1-15 ◽

Cited By ~ 42

Author(s):

Roy Jefferis

Keyword(s):

Posttranslational Modifications ◽

Drug Product ◽

Molecular Forms ◽

Phagocytic Cells ◽

Production Platform ◽

The Individual ◽

And Function ◽

Antigen Presenting

Whilst the amino acid sequence of a protein is determined by its gene sequence, the final structure and function are determined by posttranslational modifications (PTMs), including quality control (QC) in the endoplasmic reticulum (ER) and during passage through the Golgi apparatus. These processes are species and cell specific and challenge the biopharmaceutical industry when developing a production platform for the generation of recombinant biologic therapeutics. Proteins and glycoproteins are also subject to chemical modifications (CMs) bothin vivoandin vitro. The individual is naturally tolerant to molecular forms of self-molecules but nonself variants can provoke an immune response with the generation of anti-drug antibodies (ADA); aggregated forms can exhibit enhanced immunogenicity and QC procedures are developed to avoid or remove them. Monoclonal antibody therapeutics (mAbs) are a special case because their purpose is to bind the target, with the formation of immune complexes (ICs), a particular form of aggregate. Such ICs may be removed by phagocytic cells that have antigen presenting capacity. These considerations may frustrate the possibility of ameliorating the immunogenicity of mAbs by rigorous exclusion of aggregates from drug product. Alternate strategies for inducing immunosuppression or tolerance are discussed.

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Knockout of the Golgi stacking proteins GRASP55 and GRASP65 impairs Golgi structure and function

Molecular Biology of the Cell ◽

10.1091/mbc.e17-02-0112 ◽

2017 ◽

Vol 28 (21) ◽

pp. 2833-2842 ◽

Cited By ~ 47

Author(s):

Michael E. Bekier ◽

Leibin Wang ◽

Jie Li ◽

Haoran Huang ◽

Danming Tang ◽

...

Keyword(s):

Posttranslational Modifications ◽

Protein Trafficking ◽

Critical Role ◽

Hek293 Cells ◽

Double Knockout ◽

Knock Out ◽

Golgi Stack ◽

Cellular Processes ◽

Golgi Structure ◽

And Function

Golgi reassembly stacking protein of 65 kDa (GRASP65) and Golgi reassembly stacking protein of 55 kDa (GRASP55) were originally identified as Golgi stacking proteins; however, subsequent GRASP knockdown experiments yielded inconsistent results with respect to the Golgi structure, indicating a limitation of RNAi-based depletion. In this study, we have applied the recently developed clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology to knock out GRASP55 and GRASP65, individually or in combination, in HeLa and HEK293 cells. We show that double knockout of GRASP proteins disperses the Golgi stack into single cisternae and tubulovesicular structures, accelerates protein trafficking, and impairs accurate glycosylation of proteins and lipids. These results demonstrate a critical role for GRASPs in maintaining the stacked structure of the Golgi, which is required for accurate posttranslational modifications in the Golgi. Additionally, the GRASP knockout cell lines developed in this study will be useful tools for studying the role of GRASP proteins in other important cellular processes.

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C Terminus of Nce102 Determines the Structure and Function of Microdomains in the Saccharomyces cerevisiae Plasma Membrane

Eukaryotic Cell ◽

10.1128/ec.00006-10 ◽

2010 ◽

Vol 9 (8) ◽

pp. 1184-1192 ◽

Cited By ~ 24

Author(s):

Martin Loibl ◽

Guido Grossmann ◽

Vendula Stradalova ◽

Andreas Klingl ◽

Reinhard Rachel ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Plasma Membrane ◽

Protein Trafficking ◽

Ultrastructural Feature ◽

Membrane Topology ◽

Content Type ◽

C Terminus ◽

Composition And Structure ◽

And Function ◽

Arginine Permease

ABSTRACT The plasma membrane of the yeast Saccharomyces cerevisiae contains stably distributed lateral domains of specific composition and structure, termed MCC (membrane compartment of arginine permease Can1). Accumulation of Can1 and other specific proton symporters within MCC is known to regulate the turnover of these transporters and is controlled by the presence of another MCC protein, Nce102. We show that in an NCE102 deletion strain the function of Nce102 in directing the specific permeases into MCC can be complemented by overexpression of the NCE102 close homolog FHN1 (the previously uncharacterized YGR131W) as well as by distant Schizosaccharomyces pombe homolog fhn1 (SPBC1685.13). We conclude that this mechanism of plasma membrane organization is conserved through the phylum Ascomycota. We used a hemagglutinin (HA)/Suc2/His4C reporter to determine the membrane topology of Nce102. In contrast to predictions, its N and C termini are oriented toward the cytosol. Deletion of the C terminus or even of its last 6 amino acids does not disturb protein trafficking, but it seriously affects the formation of MCC. We show that the C-terminal part of the Nce102 protein is necessary for localization of both Nce102 itself and Can1 to MCC and also for the formation of furrow-like membrane invaginations, the characteristic ultrastructural feature of MCC domains.

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Platelet fibrinogen and vitronectin in Glanzmann thrombasthenia: evidence consistent with specific roles for glycoprotein IIb/IIIA and alpha v beta 3 integrins in platelet protein trafficking [see comments]

Blood ◽

10.1182/blood.v78.10.2603.2603 ◽

1991 ◽

Vol 78 (10) ◽

pp. 2603-2610 ◽

Cited By ~ 49

Author(s):

BS Coller ◽

U Seligsohn ◽

SM West ◽

LE Scudder ◽

KJ Norton

Keyword(s):

Protein Trafficking ◽

Extracellular Proteins ◽

Platelet Glycoprotein ◽

Integrin Receptors ◽

Vitronectin Receptor ◽

Glanzmann Thrombasthenia ◽

Platelet Protein ◽

Individual Contributions ◽

The Individual ◽

Alpha V Beta 3

Abstract To assess the individual contributions of the platelet glycoprotein (GP) IIb/IIIa receptor and the alpha v beta 3 vitronectin receptor to platelet levels of fibrinogen and vitronectin, we analyzed the platelets from two groups of Glanzmann thrombasthenic patients: Iraqi- Jews, whose platelets lack both receptors, and Arab patients in Israel, whose platelets lack GPIIb/IIIa, but have normal or increased numbers of alpha v beta 3 vitronectin receptors. The platelets from both thrombasthenic groups had profound deficiencies of fibrinogen, but the defect in the Iraqi-Jewish patients' platelets appeared to be slightly more severe. This finding indicates that GPIIb/IIIa is the major determinant of platelet fibrinogen, presumably acting by receptor- mediated uptake, and that the alpha v beta 3 vitronectin receptor plays little or no role. Arab patients' platelets have normal amounts of platelet vitronectin, whereas Iraqi-Jewish patients' platelets have nearly five times as much vitronectin as control or Arab patients' platelets. To account for these data, we propose a working hypothesis in which vitronectin is synthesized in megakaryocytes and the alpha v beta 3 vitronectin receptor is involved in transport of the protein out of megakaryocytes and/or platelets. Collectively, these observations suggest that in addition to their recognized roles in cell adhesion and in the interaction of cells with extracellular proteins, integrin receptors may be important in protein trafficking into, and perhaps out of, platelets.

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Platelet fibrinogen and vitronectin in Glanzmann thrombasthenia: evidence consistent with specific roles for glycoprotein IIb/IIIA and alpha v beta 3 integrins in platelet protein trafficking [see comments]

Blood ◽

10.1182/blood.v78.10.2603.bloodjournal78102603 ◽

1991 ◽

Vol 78 (10) ◽

pp. 2603-2610 ◽

Cited By ~ 3

Author(s):

BS Coller ◽

U Seligsohn ◽

SM West ◽

LE Scudder ◽

KJ Norton

Keyword(s):

Protein Trafficking ◽

Extracellular Proteins ◽

Platelet Glycoprotein ◽

Integrin Receptors ◽

Vitronectin Receptor ◽

Glanzmann Thrombasthenia ◽

Platelet Protein ◽

Individual Contributions ◽

The Individual ◽

Alpha V Beta 3

To assess the individual contributions of the platelet glycoprotein (GP) IIb/IIIa receptor and the alpha v beta 3 vitronectin receptor to platelet levels of fibrinogen and vitronectin, we analyzed the platelets from two groups of Glanzmann thrombasthenic patients: Iraqi- Jews, whose platelets lack both receptors, and Arab patients in Israel, whose platelets lack GPIIb/IIIa, but have normal or increased numbers of alpha v beta 3 vitronectin receptors. The platelets from both thrombasthenic groups had profound deficiencies of fibrinogen, but the defect in the Iraqi-Jewish patients' platelets appeared to be slightly more severe. This finding indicates that GPIIb/IIIa is the major determinant of platelet fibrinogen, presumably acting by receptor- mediated uptake, and that the alpha v beta 3 vitronectin receptor plays little or no role. Arab patients' platelets have normal amounts of platelet vitronectin, whereas Iraqi-Jewish patients' platelets have nearly five times as much vitronectin as control or Arab patients' platelets. To account for these data, we propose a working hypothesis in which vitronectin is synthesized in megakaryocytes and the alpha v beta 3 vitronectin receptor is involved in transport of the protein out of megakaryocytes and/or platelets. Collectively, these observations suggest that in addition to their recognized roles in cell adhesion and in the interaction of cells with extracellular proteins, integrin receptors may be important in protein trafficking into, and perhaps out of, platelets.

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Endoproteolytic Processing of RhoA by Rce1 Is Required for the Cleavage of RhoA by Yersinia enterocolitica Outer Protein T

Infection and Immunity ◽

10.1128/iai.74.3.1712-1717.2006 ◽

2006 ◽

Vol 74 (3) ◽

pp. 1712-1717 ◽

Cited By ~ 12

Author(s):

Florian Fueller ◽

Martin O. Bergo ◽

Stephen G. Young ◽

Klaus Aktories ◽

Gudula Schmidt

Keyword(s):

Amino Acids ◽

Proteolytic Activity ◽

Rho Gtpases ◽

Mammalian Cells ◽

Living Cells ◽

Type I ◽

Rho Proteins ◽

Carboxyl Methylation ◽

Carboxyl Terminal ◽

Caax Motif

ABSTRACT The bacterial toxin Yersinia outer protein T (YopT) is a cysteine protease that cleaves Rho GTPases immediately upstream of a carboxyl-terminal isoprenylcysteine. By clipping off the lipid anchor, YopT releases Rho GTPases from membranes, resulting in rounding up of mammalian cells in culture. The proteolytic activity of YopT depends on the isoprenylation of the cysteine within the carboxyl-terminal CaaX motif, a reaction carried out by geranylgeranyltransferase type I. The CaaX motif (where “a” indicates aliphatic amino acids) of Rho proteins undergoes two additional processing steps: endoproteolytic removal of the last three amino acids (i.e., -aaX) by Rce1 (Ras-converting enzyme 1) and methylation of the geranylgeranylcysteine by Icmt (isoprenylcysteine carboxyl methyltransferase). In in vitro experiments, RhoA retaining -aaX cannot be cleaved by YopT. Nothing is known, however, about the influence of Rce1-mediated removal of -aaX on the activity of YopT in living cells. We hypothesized that Rce1-deficient mouse fibroblasts, in which the geranylgeranylated Rho proteins are not endoproteolytically processed, would be resistant to YopT. Indeed, this was the case. Microinjection of recombinant YopT into Rce1-deficient fibroblasts had no impact on the subcellular localization of RhoA and no impact on cell morphology. To determine if carboxyl methylation is also required for YopT action, we microinjected YopT into Icmt-deficient fibroblasts. In contrast to the results with Rce1-deficient cells, YopT cleaved RhoA and caused rounding up of the Icmt-deficient cells. Our data demonstrate that Rce1-mediated removal of -aaX from isoprenylated Rho GTPases is required for the proteolytic activity of YopT in living cells, whereas carboxyl methylation by Icmt is not.

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Two structurally distinct domains of the nucleoporin Nup170 cooperate to tether a subset of nucleoporins to nuclear pores

The Journal of Cell Biology ◽

10.1083/jcb.200810016 ◽

2009 ◽

Vol 185 (3) ◽

pp. 387-395 ◽

Cited By ~ 22

Author(s):

Dirk Flemming ◽

Phillip Sarges ◽

Philipp Stelter ◽

Andrea Hellwig ◽

Bettina Böttcher ◽

...

Keyword(s):

Pore Structure ◽

Structure And Function ◽

Nuclear Pore ◽

Nuclear Pores ◽

Terminal Domain ◽

C Terminus ◽

N Terminus ◽

The Individual ◽

And Function

How individual nucleoporins (Nups) perform their role in nuclear pore structure and function is largely unknown. In this study, we examined the structure of purified Nup170 to obtain clues about its function. We show that Nup170 adopts a crescent moon shape with two structurally distinct and separable domains, a β-propeller N terminus and an α-solenoid C terminus. To address the individual roles of each domain, we expressed these domains separately in yeast. Notably, overexpression of the Nup170 C domain was toxic in nup170Δ cells and caused accumulation of several Nups in cytoplasmic foci. Further experiments indicated that the C-terminal domain anchors Nup170 to nuclear pores, whereas the N-terminal domain functions to recruit or retain a subset of Nups, including Nup159, Nup188, and Pom34, at nuclear pores. We conclude that Nup170 performs its role as a structural adapter between cytoplasmically oriented Nups and the nuclear pore membrane.

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The C-terminal GGAP motif of Hsp70 mediates substrate recognition and stress response in yeast

Journal of Biological Chemistry ◽

10.1074/jbc.ra118.002691 ◽

2018 ◽

Vol 293 (46) ◽

pp. 17663-17675 ◽

Cited By ~ 8

Author(s):

Weibin Gong ◽

Wanhui Hu ◽

Linan Xu ◽

Huiwen Wu ◽

Si Wu ◽

...

Keyword(s):

Substrate Binding ◽

Substrate Recognition ◽

Fibril Formation ◽

Helical Bundle ◽

Chaperone Function ◽

Binding Domains ◽

C Terminus ◽

Individual Contributions ◽

The Individual

The allosteric coupling of the highly conserved nucleotide- and substrate-binding domains of Hsp70 has been studied intensively. In contrast, the role of the disordered, highly variable C-terminal region of Hsp70 remains unclear. In many eukaryotic Hsp70s, the extreme C-terminal EEVD motif binds to the tetratricopeptide-repeat domains of Hsp70 co-chaperones. Here, we discovered that the TVEEVD sequence of Saccharomyces cerevisiae cytoplasmic Hsp70 (Ssa1) functions as a SUMO-interacting motif. A second C-terminal motif of ∼15 amino acids between the α-helical lid and the extreme C terminus, previously identified in bacterial and eukaryotic organellar Hsp70s, is known to enhance chaperone function by transiently interacting with folding clients. Using structural analysis, interaction studies, fibril formation assays, and in vivo functional assays, we investigated the individual contributions of the α-helical bundle and the C-terminal disordered region of Ssa1 in the inhibition of fibril formation of the prion protein Ure2. Our results revealed that although the α-helical bundle of the Ssa1 substrate-binding domain (SBDα) does not directly bind to Ure2, the SBDα enhances the ability of Hsp70 to inhibit fibril formation. We found that a 20-residue C-terminal motif in Ssa1, containing GGAP and GGAP-like tetrapeptide repeats, can directly bind to Ure2, the Hsp40 co-chaperone Ydj1, and α-synuclein, but not to the SUMO-like protein SMT3 or BSA. Deletion or substitution of the Ssa1 GGAP motif impaired yeast cell tolerance to temperature and cell-wall damage stress. This study highlights that the C-terminal GGAP motif of Hsp70 is important for substrate recognition and mediation of the heat shock response.

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NRAS is unique among RAS proteins in requiring ICMT for trafficking to the plasma membrane

Life Science Alliance ◽

10.26508/lsa.202000972 ◽

2021 ◽

Vol 4 (5) ◽

pp. e202000972

Author(s):

Ian M Ahearn ◽

Helen R Court ◽

Farid Siddiqui ◽

Daniel Abankwa ◽

Mark R Philips

Keyword(s):

Plasma Membrane ◽

Ras Proteins ◽

The Third ◽

Carboxyl Methylation ◽

Hypervariable Regions ◽

Carboxyl Methyltransferase ◽

C Terminus ◽

Direct Delivery ◽

Protein Chaperone ◽

Palmitoylation Site

Isoprenylcysteine carboxyl methyltransferase (ICMT) is the third of three enzymes that sequentially modify the C-terminus of CaaX proteins, including RAS. Although all four RAS proteins are substrates for ICMT, each traffics to membranes differently by virtue of their hypervariable regions that are differentially palmitoylated. We found that among RAS proteins, NRAS was unique in requiring ICMT for delivery to the PM, a consequence of having only a single palmitoylation site as its secondary affinity module. Although not absolutely required for palmitoylation, acylation was diminished in the absence of ICMT. Photoactivation and FRAP of GFP-NRAS revealed increase flux at the Golgi, independent of palmitoylation, in the absence of ICMT. Association of NRAS with the prenyl-protein chaperone PDE6δ also required ICMT and promoted anterograde trafficking from the Golgi. We conclude that carboxyl methylation of NRAS is required for efficient palmitoylation, PDE6δ binding, and homeostatic flux through the Golgi, processes that direct delivery to the plasma membrane.

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Narrow is the Gate to Utopia

Moreana ◽

10.3366/more.2012.49.1-2.11 ◽

2012 ◽

Vol 49 (Number 187- (1-2) ◽

pp. 207-226

Author(s):

Marie-Claire Phélippeau

Keyword(s):

Social Construction ◽

Free Will ◽

Religious Toleration ◽

Individual Liberty ◽

The Reformation ◽

The Individual ◽

And Function

This study examines the notions of pleasure, individual liberty and consensus in Thomas More’s Utopia. The paradox inherent in Utopia, written before the Reformation, is especially visible in the affirmation of religious toleration coexisting with the need for a strict supervision of the citizens. The dream of an ideal republic is based on a Pauline vision of man which defines the individual mainly as a sinner. Consequently, it is the duty of the republic’s rulers to guide the citizens and establish a consensus. This study tries to determine the part left to the individual’s free will and examines the nature and function of the structures that are supposed to ensure the happiness of each one and of the whole community. The notion of moral hierarchy is asserted as the linchpin of the Utopian social construction.

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