scholarly journals Palmitoylation of Sindbis Virus TF Protein Regulates Its Plasma Membrane Localization and Subsequent Incorporation into Virions

2016 ◽  
Vol 91 (3) ◽  
Author(s):  
Jolene Ramsey ◽  
Emily C. Renzi ◽  
Randy J. Arnold ◽  
Jonathan C. Trinidad ◽  
Suchetana Mukhopadhyay
Keyword(s):  
Plasma Membrane ◽  
Sindbis Virus ◽  
Molecular Mechanisms ◽  
Wild Type Virus ◽  
Membrane Localization ◽  
Clear Understanding ◽  
Wild Type ◽  
Plasma Membrane Localization ◽  
C Terminus ◽  
N Terminus

ABSTRACT Palmitoylation is a reversible, posttranslational modification that helps target proteins to cellular membranes. The alphavirus small membrane proteins 6K and TF have been reported to be palmitoylated and to positively regulate budding. 6K and TF are isoforms that are identical in their N termini but unique in their C termini due to a −1 ribosomal frameshift during translation. In this study, we used cysteine (Cys) mutants to test differential palmitoylation of the Sindbis virus 6K and TF proteins. We modularly mutated the five Cys residues in the identical N termini of 6K and TF, the four additional Cys residues in TF's unique C terminus, or all nine Cys residues in TF. Using these mutants, we determined that TF palmitoylation occurs primarily in the N terminus. In contrast, 6K is not palmitoylated, even on these shared residues. In the C-terminal Cys mutant, TF protein levels increase both in the cell and in the released virion compared to the wild type. In viruses with the N-terminal Cys residues mutated, TF is much less efficiently localized to the plasma membrane, and it is not incorporated into the virion. The three Cys mutants have minor defects in cell culture growth but a high incidence of abnormal particle morphologies compared to the wild-type virus as determined by transmission electron microscopy. We propose a model where the C terminus of TF modulates the palmitoylation of TF at the N terminus, and palmitoylated TF is preferentially trafficked to the plasma membrane for virus budding. IMPORTANCE Alphaviruses are a reemerging viral cause of arthritogenic disease. Recently, the small 6K and TF proteins of alphaviruses were shown to contribute to virulence in vivo. Nevertheless, a clear understanding of the molecular mechanisms by which either protein acts to promote virus infection is missing. The TF protein is a component of budded virions, and optimal levels of TF correlate positively with wild-type-like particle morphology. In this study, we show that the palmitoylation of TF regulates its localization to the plasma membrane, which is the site of alphavirus budding. Mutants in which TF is not palmitoylated display drastically reduced plasma membrane localization, which effectively prevents TF from participating in budding or being incorporated into virus particles. Investigation of the regulation of TF will aid current efforts in the alphavirus field searching for approaches to mitigate alphaviral disease in humans.

scholarly journals Modification of the 5′ Terminus of Sindbis Virus Genomic RNA Allows nsP4 RNA Polymerases with Nonaromatic Amino Acids at the N Terminus To Function in RNA Replication

2003 ◽  
Vol 77 (4) ◽  
pp. 2301-2309 ◽  
Author(s):  
Yukio Shirako ◽  
Ellen G. Strauss ◽  
James H. Strauss
Keyword(s):  
Amino Acid ◽  
Secondary Structure ◽  
Sindbis Virus ◽  
Rna Synthesis ◽  
Wild Type Virus ◽  
Progeny Virus ◽  
Minus Strand ◽  
Genomic Rna ◽  
Wild Type ◽  
N Terminus

ABSTRACT We have previously shown that Sindbis virus RNA polymerase requires an N-terminal aromatic amino acid or histidine for wild-type or pseudo-wild-type function; mutant viruses with a nonaromatic amino acid at the N terminus of the polymerase, but which are otherwise wild type, are unable to produce progeny viruses and will not form a plaque at any temperature tested. We now show that such mutant polymerases can function to produce progeny virus sufficient to form plaques at both 30 and 40°C upon addition of AU, AUA, or AUU to the 5′ terminus of the genomic RNA or upon substitution of A for U as the third nucleotide of the genome. These results are consistent with the hypothesis that (i) 3′-UA-5′ is required at the 3′ terminus of the minus-strand RNA for initiation of plus-strand genomic RNA synthesis; (ii) in the wild-type virus this sequence is present in a secondary structure that can be opened by the wild-type polymerase but not by the mutant polymerase; (iii) the addition of AU, AUA, or AUU to the 5′ end of the genomic RNA provides unpaired 3′-UA-5′ at the 3′ end of the minus strand that can be utilized by the mutant polymerase, and similarly, the effect of the U3A mutation is to destabilize the secondary structure, freeing 3′-terminal UA; and (iv) the N terminus of nsP4 may directly interact with the 3′ terminus of the minus-strand RNA for the initiation of the plus-strand genomic RNA synthesis. This hypothesis is discussed in light of our present results as well as of previous studies of alphavirus RNAs, including defective interfering RNAs.

scholarly journals Plasma membrane-targeted ras GTPase-activating protein is a potent suppressor of p21ras function.

1993 ◽  
Vol 13 (4) ◽  
pp. 2420-2431 ◽  
Author(s):  
D C Huang ◽  
C J Marshall ◽  
J F Hancock
Keyword(s):  
Plasma Membrane ◽  
Catalytic Activity ◽  
Catalytic Domain ◽  
Cell Transformation ◽  
Negative Regulator ◽  
Membrane Localization ◽  
Plasma Membrane Localization ◽  
Terminal Domain ◽  
3T3 Fibroblasts ◽  
C Terminus

Although p21ras is localized to the plasma membrane, proteins it interacts with, such as the GTPase-activating proteins (GAPs) ras GAP and neurofibromin (NF1), are not, suggesting that one function of p21ras GTP may be to target such proteins to the plasma membrane. To investigate the effects of targeting ras GAP to the plasma membrane, ras C-terminal motifs sufficient for plasma membrane localization of p21ras were cloned onto the C terminus of ras GAP. Plasma membrane-targeted ras GAP is growth inhibitory to NIH 3T3 fibroblasts and COS cells. This growth inhibition correlates with GAP catalytic activity, since the plasma membrane-targeted C-terminal catalytic domain or the GAP-related domain of neurofibromin is inhibitory, whereas the similarly targeted N-terminal domain is not. Moreover, the inhibition is abrogated by the inactivating mutation L902I, which abolishes ras GAP catalytic activity. Coexpression of oncogenic mutant ras rescues cell viability, but the majority of rescued colonies are phenotypically untransformed. Furthermore, in focus assays, targeted ras GAP suppresses transformation by oncogenic mutant ras, and in reversion assays, targeted ras GAP can revert cells transformed by oncogenic mutant ras. Neither the targeted or nontargeted N-terminal domain nor the L902I mutant of ras GAP has any transforming activity. These data demonstrate that ras GAP can function as a negative regulator of ras and that plasma membrane localization potentiates this activity. However, if ras GAP is involved in the effector functions of p21ras, it can only be part of the effector complex for cell transformation.

scholarly journals Requirement for an Aromatic Amino Acid or Histidine at the N Terminus of Sindbis Virus RNA Polymerase

1998 ◽  
Vol 72 (3) ◽  
pp. 2310-2315 ◽  
Author(s):  
Yukio Shirako ◽  
James H. Strauss
Keyword(s):  
Amino Acid ◽  
Rna Polymerase ◽  
Sindbis Virus ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Prolonged Incubation ◽  
Rapid Degradation ◽  
N Terminus ◽  
Reticulocyte Lysates

ABSTRACT The N terminal amino acid of nonstructural protein nsP4, the viral RNA polymerase, is a tyrosine in all sequenced alphaviruses; this is a destabilizing amino acid for the N-end rule pathway and results in rapid degradation of nsP4 produced in infected cells or in reticulocyte lysates. We have constructed 11 mutants of Sindbis virus bearing Phe, Ala, Thr, Cys, Leu, Met, Asn, Gln, Glu, Arg, or Pro at the N terminus of nsP4. Translation of RNAs in reticulocyte lysates showed that cleavage at the nsP3/nsP4 site occurred efficiently for all mutants except for Glu-nsP4, which was cleaved inefficiently, and Pro-nsP4, which was not detectably cleaved, and that Tyr, Cys, Leu, Arg, and Phe destabilized nsP4 but Ala, Met, Thr, Asn, Gln, and Glu stabilized nsP4 to various extents. The viability of the mutants was examined by transfection of chicken cells at 30 or 40°C. The Phe-nsP4 mutant formed large plaques at both temperatures. The Met-nsP4 mutant was also viable but formed small plaques at 30°C and minute plaques at 40°C. The remaining mutants did not form plaques at either temperature. However, after prolonged incubation at 30°C, all the mutants except Glu-nsP4 and Pro-nsP4 produced viable viruses. In the case of Cys-, Leu-, Asn-, Gln-, or Arg-nsP4, revertants that were indistinguishable in plaque phenotype from the wild-type virus arose by same-site reversion to Tyr, Trp, Phe, or His by a single nucleotide substitution in the original mutant codon. Viable viruses also arose from the Ala-, Leu-, Cys-, Thr-, Asn-, Gln-, and Arg-nsP4 mutants that retained the original mutations at the N terminus of nsP4, but these viruses formed smaller plaques than the wild-type virus and many were temperature sensitive. Our results indicate that only nsP4s bearing N-terminal Tyr, Phe, Trp, or His have wild-type or near-wild-type activity for RNA replication and that rapid degradation of nsP4 is not a prerequisite for its function. nsP4s bearing other N-terminal residues, with the exception of Met-nsP4, have only very low or negligible activity, so that no detectable infectious virus can be produced. However, suppressor mutations can arise that enable most such nsP4s to regain significant but still suboptimal activity.

Plasma membrane-targeted ras GTPase-activating protein is a potent suppressor of p21ras function

1993 ◽  
Vol 13 (4) ◽  
pp. 2420-2431
Author(s):  
D C Huang ◽  
C J Marshall ◽  
J F Hancock
Keyword(s):  
Plasma Membrane ◽  
Catalytic Activity ◽  
Catalytic Domain ◽  
Cell Transformation ◽  
Negative Regulator ◽  
Membrane Localization ◽  
Plasma Membrane Localization ◽  
Terminal Domain ◽  
3T3 Fibroblasts ◽  
C Terminus

Although p21ras is localized to the plasma membrane, proteins it interacts with, such as the GTPase-activating proteins (GAPs) ras GAP and neurofibromin (NF1), are not, suggesting that one function of p21ras GTP may be to target such proteins to the plasma membrane. To investigate the effects of targeting ras GAP to the plasma membrane, ras C-terminal motifs sufficient for plasma membrane localization of p21ras were cloned onto the C terminus of ras GAP. Plasma membrane-targeted ras GAP is growth inhibitory to NIH 3T3 fibroblasts and COS cells. This growth inhibition correlates with GAP catalytic activity, since the plasma membrane-targeted C-terminal catalytic domain or the GAP-related domain of neurofibromin is inhibitory, whereas the similarly targeted N-terminal domain is not. Moreover, the inhibition is abrogated by the inactivating mutation L902I, which abolishes ras GAP catalytic activity. Coexpression of oncogenic mutant ras rescues cell viability, but the majority of rescued colonies are phenotypically untransformed. Furthermore, in focus assays, targeted ras GAP suppresses transformation by oncogenic mutant ras, and in reversion assays, targeted ras GAP can revert cells transformed by oncogenic mutant ras. Neither the targeted or nontargeted N-terminal domain nor the L902I mutant of ras GAP has any transforming activity. These data demonstrate that ras GAP can function as a negative regulator of ras and that plasma membrane localization potentiates this activity. However, if ras GAP is involved in the effector functions of p21ras, it can only be part of the effector complex for cell transformation.

Dual functional significance of calcineurin homologous protein 1 binding to Na+/H+ exchanger isoform 1

2011 ◽  
Vol 301 (2) ◽  
pp. C280-C288 ◽  
Author(s):  
Masafumi Matsushita ◽  
Hiroo Tanaka ◽  
Keiji Mitsui ◽  
Hiroshi Kanazawa
Keyword(s):  
Plasma Membrane ◽  
Binding Site ◽  
Functional Significance ◽  
Gene Knockout ◽  
Membrane Localization ◽  
Wild Type ◽  
Homologous Protein ◽  
Plasma Membrane Localization ◽  
Dual Functional ◽  
Associated Proteins

Calcineurin homologous protein 1 (CHP1) binds to the hydrophilic tail of the Na+/H+ exchanger isoform 1 (NHE1). Previous gene knockout of CHP1 revealed that the loss of CHP1 caused a decrease in the total amount of NHE1, suggesting the destabilization of NHE1 molecules without CHP1 (Matsushita et al., Am J Physiol Cell Physiol 293: C246–C254, 2007). However, Pang et al. ( J Biol Chem 276: 17367–17372, 2001) reported that NHE1 without a CHP1 binding site was found in the plasma membrane, suggesting no requirement of CHP1 binding for plasma membrane localization of NHE1. Here, the functional significance of CHP1 binding to NHE1 was examined to resolve these contradictory results. In CV1 cells, which overexpressed wild-type NHE1, overexpression of CHP1 caused an increase in both the total amount of NHE1 and the colocalization of NHE1 and CHP1 at the plasma membrane. This provided new visual evidence of the localization of NHE1 from endoplasmic reticulum to the plasma membrane upon CHP1 binding. An immunoprecipitation assay showed that the expression of CHP1 reduced the ubiquitination of NHE1 and/or its associated proteins. Mutant NHE1s without CHP1 binding site exhibited a modest localization to the plasma membrane. After reaching the plasma membrane, these mutant NHE1s exhibited shorter half-lives than the wild-type NHE1 with CHP1. The results suggest a dual functional significance of CHP1 and its binding region: 1) binding of CHP1 stabilizes NHE1 and increases its plasma membrane localization by masking a NHE1 disposal signal, and 2) CHP1 binding is required for the antiporter activity.

scholarly journals C-terminus Proteolysis and Palmitoylation Cooperate for Optimal Plasma Membrane Localization of RasA in Aspergillus fumigatus

2018 ◽  
Vol 9 ◽  
Author(s):  
Qusai Al Abdallah ◽  
Adela Martin-Vicente ◽  
Ana Camila Oliveira Souza ◽  
Wenbo Ge ◽  
Jarrod R. Fortwendel
Keyword(s):  
Plasma Membrane ◽  
Aspergillus Fumigatus ◽  
Membrane Localization ◽  
Plasma Membrane Localization ◽  
C Terminus

Protein 4.2 interaction with hereditary spherocytosis mutants of the cytoplasmic domain of human anion exchanger 1

2010 ◽  
Vol 433 (2) ◽  
pp. 313-322 ◽  
Author(s):  
Susan P. Bustos ◽  
Reinhart A. F. Reithmeier
Keyword(s):  
Plasma Membrane ◽  
Anion Exchanger ◽  
Hereditary Spherocytosis ◽  
Cytoplasmic Domain ◽  
Membrane Localization ◽  
Wild Type ◽  
Anion Exchanger 1 ◽  
Plasma Membrane Localization ◽  
293 Cells ◽  
Protein 4.2

AE1 (anion exchanger 1) and protein 4.2 associate in a protein complex bridging the erythrocyte membrane and cytoskeleton; disruption of the complex results in unstable erythrocytes and HS (hereditary spherocytosis). Three HS mutations (E40K, G130R and P327R) in cdAE1 (the cytoplasmic domain of AE1) occur with deficiencies of protein 4.2. The interaction of wild-type AE1, AE1HS mutants, mdEA1 (the membrane domain of AE1), kAE1 (the kidney isoform of AE1) and AE1SAO (Southeast Asian ovalocytosis AE1) with protein 4.2 was examined in transfected HEK (human embryonic kidney)-293 cells. The HS mutants had wild-type expression levels and plasma-membrane localization. Protein 4.2 expression was not dependent on AE1. Protein 4.2 was localized throughout the cytoplasm and co-localized at the plasma membrane with the HS mutants mdAE1 and kAE1, but at the ER (endoplasmic reticulum) with AE1SAO. Pull-down assays revealed diminished levels of protein 4.2 associated with the HS mutants relative to AE1. The mdAE1 did not bind protein 4.2, whereas kAE1 and AE1SAO bound wild-type amounts of protein 4.2. A protein 4.2 fatty acylation mutant, G2A/C173A, had decreased plasma-membrane localization compared with wild-type protein 4.2, and co-expression with AE1 enhanced its plasma-membrane localization. Subcellular fractionation showed the majority of wild-type and G2A/C173A protein 4.2 was associated with the cytoskeleton of HEK-293 cells. The present study shows that cytoplasmic HS mutants cause impaired binding of protein 4.2 to AE1, leaving protein 4.2 susceptible to loss during erythrocyte development.

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