Characterization of a novel phosphatidylinositol 3-phosphate-binding protein containing two FYVE fingers in tandem that is targeted to the Golgi

2001 ◽  
Vol 355 (1) ◽  
pp. 113-121 ◽  
Author(s):  
Peter C. F. CHEUNG ◽  
Laura TRINKLE-MULCAHY ◽  
Philip COHEN ◽  
John M. LUCOCQ
Keyword(s):  
Fluorescent Protein ◽  
Mutational Analysis ◽  
Consensus Sequence ◽  
Phosphate Binding ◽  
Protein Fusion ◽  
Cell Localization ◽  
Phosphate Binding Protein ◽  
Green Fluorescent ◽  
Novel Protein

We have identified a novel protein of predicted molecular mass 40kDa that contains two FYVE domains in tandem and has therefore been named TAFF1 (TAndem FYVE Fingers-1). The protein is expressed predominantly in heart and binds to PtdIns3P specifically, even though the FYVE domains in TAFF1 lacks the first Arg of the consensus sequence R(K/R)HHCR, critical for the PtdIns3P binding of other FYVE domains identified so far. The first Arg is replaced by a Thr and Ser in the N-terminal and C-terminal FYVE domains of TAFF1 respectively. Mutational analysis indicates that both FYVE domains are required for high affinity binding to PtdIns3P. Cell localization studies using a green fluorescent protein fusion show that TAFF1 is localized to the Golgi, and that the Golgi targeting sequence is located within the N-terminal 187 residues and not in either FYVE domain. The sequence of TAFF1has been deposited with the GenBank®, EMBL, DDBJ and GSDB Nucleotide Sequence Databases under accession number AF311602.

scholarly journals Functional Characterization of a Drought-Responsive Invertase Inhibitor from Maize (Zea mays L.)

2019 ◽  
Vol 20 (17) ◽  
pp. 4081 ◽  
Author(s):  
Lin Chen ◽  
Xiaohong Liu ◽  
Xiaojia Huang ◽  
Wei Luo ◽  
Yuming Long ◽  
...  
Keyword(s):  
Cell Wall ◽  
Fluorescent Protein ◽  
Functional Characterization ◽  
Protein Fusion ◽  
Drought Treatment ◽  
Vacuolar Invertase ◽  
Maize Leaves ◽  
Green Fluorescent ◽  
Proteinaceous Inhibitor

Invertases (INVs) play essential roles in plant growth in response to environmental cues. Previous work showed that plant invertases can be post-translationally regulated by small protein inhibitors (INVINHs). Here, this study characterizes a proteinaceous inhibitor of INVs in maize (Zm-INVINH4). A functional analysis of the recombinant Zm-INVINH4 protein revealed that it inhibited both cell wall and vacuolar invertase activities from maize leaves. A Zm-INVINH4::green fluorescent protein fusion experiment indicated that this protein localized in the apoplast. Transcript analysis showed that Zm-INVINH4 is specifically expressed in maize sink tissues, such as the base part of the leaves and young kernels. Moreover, drought stress perturbation significantly induced Zm-INVINH4 expression, which was accompanied with a decrease of cell wall invertase (CWI) activities and an increase of sucrose accumulation in both base parts of the leaves 2 to 7 days after pollinated kernels. In summary, the results support the hypothesis that INV-related sink growth in response to drought treatment is (partially) caused by a silencing of INV activity via drought-induced induction of Zm-INVINH4 protein.

scholarly journals Characterization of sarR, a Modulator ofsar Expression in Staphylococcus aureus

2001 ◽  
Vol 69 (2) ◽  
pp. 885-896 ◽  
Author(s):  
Adhar Manna ◽  
Ambrose L. Cheung
Keyword(s):  
Staphylococcus Aureus ◽  
Fluorescent Protein ◽  
Regulatory Protein ◽  
Virulence Determinants ◽  
Protein Fusion ◽  
Putative Gene ◽  
P2 Promoter ◽  
Green Fluorescent ◽  
Overlapping Transcripts

ABSTRACT The expression of virulence determinants in Staphylococcus aureus is controlled by global regulatory loci (e.g.,sar and agr). The sar locus is composed of three overlapping transcripts (sar P1, P3, and P2 transcripts from P1, P3, and P2 promoters, respectively), all encoding the 372-bp sarA gene. The level of SarA, the major regulatory protein, is partially controlled by the differential activation of sar promoters. We previously partially purified a ∼12 kDa protein with a DNA-specific column containing asar P2 promoter fragment. In this study, the putative gene, designated sarR, was identified and found to encode a 13.6-kDa protein with homology to SarA. Transcriptional and immunoblot studies revealed the sarR gene to be expressed in other staphylococcal strains. Recombinant SarR protein bound sarP1, P2, and P3 promoter fragments in gel shift and footprinting assays. A sarR mutant expressed a higher level of P1 transcript than the parent, as confirmed by promoter green fluorescent protein fusion assays. As the P1 transcript is the predominant sartranscript, we confirmed that the sarR mutant expressed more SarA than the parental strain. We thus proposed that SarR is a regulatory protein that binds to the sar promoters to down-regulate P1 transcription and the ensuing SarA protein expression.

scholarly journals Characterization of a Serine Proteinase Mediating Encystation of Acanthamoeba

2008 ◽  
Vol 7 (9) ◽  
pp. 1513-1517 ◽  
Author(s):  
Eun-Kyung Moon ◽  
Dong-Il Chung ◽  
Yeon-Chul Hong ◽  
Hyun-Hee Kong
Keyword(s):  
Fluorescent Protein ◽  
Serine Proteinase ◽  
Tissue Destruction ◽  
Protein Fusion ◽  
Granulomatous Amoebic Encephalitis ◽  
Causative Agents ◽  
Amoebic Keratitis ◽  
Green Fluorescent ◽  
Interfering Rna

ABSTRACT Members of the genus Acanthamoeba, amphizoic protozoan parasites, are causative agents of granulomatous amoebic encephalitis and amoebic keratitis. Proteinases play a role in various biologic actions in Acanthamoeba, including host tissue destruction, pathogenesis, and digestion of phagocytosed food. Interestingly, we found that encystation of Acanthamoeba was inhibited by the serine proteinase inhibitor phenylmethanesulfonyl fluoride. In this study, we characterize a serine proteinase that is involved in mediating the encystation of Acanthamoeba. This encystation-mediating serine proteinase (EMSP) is shown to be highly expressed during encystation by real-time PCR and Western blot analysis. Chemically synthesized small interfering RNA against EMSP inhibited the expression of EMSP mRNA and significantly reduced the encystation efficiency of Acanthamoeba. An EMSP-enhanced green fluorescent protein fusion protein localized to vesicle-like structures within the amoeba. Using LysoTracker analysis, these vesicular structures were confirmed to be lysosomes. After incubation of the transfected amoeba in encystment media, small fluorescent vesicle-like structures gathered and formed ball-like structures, which were identified as colocalizing with the autophagosome. Taken together, these results indicate that EMSP plays an important role in the differentiation of Acanthamoeba by promoting autolysis.

scholarly journals A Novel Protein Com1 Is Required for Normal Conidium Morphology and Full Virulence in Magnaporthe oryzae

2010 ◽  
Vol 23 (1) ◽  
pp. 112-123 ◽  
Author(s):  
Jun Yang ◽  
Xiaoyan Zhao ◽  
Jing Sun ◽  
Zhensheng Kang ◽  
Shengli Ding ◽  
...  
Keyword(s):  
Magnaporthe Oryzae ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Protein Fusion ◽  
Transcription Regulator ◽  
Helix Loop Helix ◽  
Localization Signal ◽  
Conidial Development ◽  
Green Fluorescent ◽  
Novel Protein

In Magnaporthe oryzae, pyriform conidia are the primary inoculum and the main source for disease dissemination in the field. In this study, we identified and characterized the COM1 gene that was disrupted in three insertional mutants producing slender conidia. COM1 encodes a putative transcription regulator unique to filamentous ascomycetes. The com1 disruption and deletion mutants had similar defects in conidium morphology and were significantly reduced in virulence on rice and barley seedlings. Microscopic examination revealed that the Δcom1 mutants were defective in appressorium turgor generation, penetration, and infectious growth. COM1 was expressed constitutively in M. oryzae. The Com1 protein had putative helix-loop-helix structures and three predicted nuclear localization signal sequences. In transformants expressing COM1335-613–enhanced green fluorescent protein fusion constructs, fluorescence signals were observed in the nucleus. Our data indicated that the COM1 gene may encode a novel transcription regulator that regulates conidial development and invasive growth in M. oryzae.

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