The Effect of the Anticipated Nuclear Localization Sequence of ‘Candidatus Phytoplasma mali’ SAP11-like Protein on Localization of the Protein and Destabilization of TCP Transcription Factor

SAP11 is an effector protein that has been identified in various phytoplasma species. It localizes in the plant nucleus and can bind and destabilize TEOSINE BRANCHES/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) transcription factors. Although SAP11 of different phytoplasma species share similar activities, their protein sequences differ greatly. Here, we demonstrate that the SAP11-like protein of ‘Candidatus Phytoplasma mali’ (‘Ca. P. mali’) strain PM19 localizes into the plant nucleus without requiring the anticipated nuclear localization sequence (NLS). We show that the protein induces crinkled leaves and siliques, and witches’ broom symptoms, in transgenic Arabidopsis thaliana (A. thaliana) plants and binds to six members of class I and all members of class II TCP transcription factors of A. thaliana in yeast two-hybrid assays. We also identified a 17 amino acid stretch previously predicted to be a nuclear localization sequence that is important for the binding of some of the TCPs, which results in a crinkled leaf and silique phenotype in transgenic A. thaliana. Moreover, we provide evidence that the SAP11-like protein has a destabilizing effect on some TCPs in vivo.

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The anticipated potential nuclear localization sequence of ‘Candidatus Phytoplasma mali’ SAP11-like protein is required for TCP binding but not for transport into the nucleus

10.1101/2020.05.13.094045 ◽

2020 ◽

Author(s):

Alisa Strohmayer ◽

Timothy Schwarz ◽

Mario Braun ◽

Gabi Krczal ◽

Kajohn Boonrod

Keyword(s):

Plant Pathogens ◽

Leader Sequence ◽

Effector Proteins ◽

Nuclear Localization Sequence ◽

Candidatus Phytoplasma Mali ◽

Localization Sequence ◽

Amino Acid Stretch ◽

Apple Production ◽

Proliferating Cell ◽

Heavy Damage

AbstractThe plant pathogen ‘Candidatus Phytoplasma mali’ (‘Ca. P. mali’) is the causing agent of apple proliferation that leads to heavy damage in apple production all over Europe. To identify and analyze effector proteins of plant pathogens is an important strategy in plant disease research. Here, we report that the SAP11-like protein of ‘Ca. P. mali’ induces crinkled leaves and siliques and witches’ broom symptoms in transgenic Arabidopsis thaliana (A. thaliana) plants and binds to 6 members of class I and all members of class II TCP (TEOSINE BRANCHES/ CYCLOIDEA/PROLIFERATING CELL FACTOR) transcription factors of A. thaliana in yeast two-hybrid assays. Moreover, we demonstrate that the protein localizes actively into the plant nucleus without requiring the nuclear leader sequence (NLS). We also identified a 17 amino acid stretch previously predicted to be a nuclear leader sequence that is important for the binding of some of the TCPs and also responsible for the crinkled leaf and silique phenotype in transgenic A. thaliana.

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Domain necessary for Drosophila ELAV nuclear localization: function requires nuclear ELAV

Journal of Cell Science ◽

10.1242/jcs.112.24.4501 ◽

1999 ◽

Vol 112 (24) ◽

pp. 4501-4512 ◽

Cited By ~ 1

Author(s):

Y.M. Yannoni ◽

K. White

Keyword(s):

Nuclear Localization ◽

Rna Binding ◽

Rna Binding Proteins ◽

Nuclear Localization Sequence ◽

Rna Recognition ◽

Rna Recognition Motifs ◽

Mutant Proteins ◽

Localization Sequence ◽

Recognition Motifs

The neuron specific Drosophila ELAV protein belongs to the ELAV family of RNA binding proteins which are characterized by three highly conserved RNA recognition motifs, an N-terminal domain, and a hinge region between the second and third RNA recognition motifs. Despite their highly conserved RNA recognition motifs the ELAV family members are a group of proteins with diverse posttranscriptional functions including splicing regulation, mRNA stability and translatability and have a variety of subcellular localizations. The role of the ELAV hinge in localization and function was examined using transgenes encoding ELAV hinge deletions, in vivo. Subcellular localization of the hinge mutant proteins revealed that residues between amino acids 333–374 are necessary for nuclear localization. This delineated sequence has no significant homology to classical nuclear localization sequences, but it is similar to the recently characterized nucleocytoplasmic shuttling sequence, the HNS, from a human ELAV family member, HuR. This defined sequence, however, was insufficient for nuclear localization as tested using hinge-GFP fusion proteins. Functional assays revealed that mutant proteins that fail to localize to the nucleus are unable to provide ELAV vital function, but their function is significantly restored when translocated into the nucleus by a heterologous nuclear localization sequence tag.

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SCCRO (DCUN1D1) Promotes Nuclear Translocation and Assembly of the Neddylation E3 Complex

Journal of Biological Chemistry ◽

10.1074/jbc.m110.203729 ◽

2011 ◽

Vol 286 (12) ◽

pp. 10297-10304 ◽

Cited By ~ 31

Author(s):

Guochang Huang ◽

Andrew J. Kaufman ◽

Y. Ramanathan ◽

Bhuvanesh Singh

Keyword(s):

Nuclear Localization ◽

Essential Role ◽

Nuclear Translocation ◽

Nuclear Localization Sequence ◽

Mouse Embryonic Fibroblasts ◽

Embryonic Fibroblasts ◽

Transgenic Expression ◽

Localization Sequence ◽

Efficient Transfer

SCCRO/DCUN1D1/DCN1 (squamous cell carcinoma-related oncogene/defective in cullin neddylation 1 domain containing 1/defective in cullin neddylation) serves as an accessory E3 in neddylation by binding to cullin and Ubc12 to allow efficient transfer of Nedd8. In this work we show that SCCRO has broader, pleiotropic effects that are essential for cullin neddylation in vivo. Reduced primary nuclear localization of Cul1 accompanying decreased neddylation and proliferation in SCCRO−/− mouse embryonic fibroblasts led us to investigate whether compartmentalization plays a regulatory role. Decreased nuclear localization, neddylation, and defective proliferation in SCCRO−/− mouse embryonic fibroblasts were rescued by transgenic expression of SCCRO. Expression of reciprocal SCCRO and Cul1-binding mutants confirmed the requirement for SCCRO in nuclear translocation and neddylation of cullins in vivo. Nuclear translocation of Cul1 by tagging with a nuclear localization sequence allowed neddylation independent of SCCRO, but at a lower level. We found that in the nucleus, SCCRO enhances recruitment of Ubc12 to Cul1 to promote neddylation. These findings suggest that SCCRO has an essential role in neddylation in vivo involving nuclear localization of neddylation components and recruitment and proper positioning of Ubc12.

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The Unfolded Protein Response Transducer Ire1p Contains a Nuclear Localization Sequence Recognized by Multiple β Importins

Molecular Biology of the Cell ◽

10.1091/mbc.e06-04-0292 ◽

2006 ◽

Vol 17 (12) ◽

pp. 5309-5323 ◽

Cited By ~ 18

Author(s):

Laurence Goffin ◽

Sadanand Vodala ◽

Christine Fraser ◽

Joanne Ryan ◽

Mark Timms ◽

...

Keyword(s):

Unfolded Protein Response ◽

Nuclear Localization ◽

Nuclear Localization Sequence ◽

Binding Studies ◽

Linker Region ◽

Unfolded Protein ◽

Localization Sequence ◽

The Unfolded Protein Response ◽

Protein Response

The Ire1p transmembrane receptor kinase/endonuclease transduces the unfolded protein response (UPR) from the endoplasmic reticulum (ER) to the nucleus in Saccharomyces cerevisiae. In this study, we analyzed the capacity of a highly basic sequence in the linker region of Ire1p to function as a nuclear localization sequence (NLS) both in vivo and in vitro. This 18-residue sequence is capable of targeting green fluorescent protein to the nucleus of yeast cells in a process requiring proteins involved in the Ran GTPase cycle that facilitates nuclear import. Mutagenic analysis and importin binding studies demonstrate that the Ire1p linker region contains overlapping potential NLSs: at least one classical NLS (within sequences 642KKKRKR647 and/or 653KKGR656) that is recognized by yeast importin α (Kap60p) and a novel βNLS (646KRGSRGGKKGRK657) that is recognized by several yeast importin β homologues. Kinetic binding data suggest that binding to importin β proteins would predominate in vivo. The UPR, and in particular ER stress-induced HAC1 mRNA splicing, is inhibited by point mutations in the Ire1p NLS that inhibit nuclear localization and also requires functional RanGAP and Ran GEF proteins. The NLS-dependent nuclear localization of Ire1p would thus seem to be central to its role in UPR signaling.

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Insights into the cellular mechanism of the yeast ubiquitin ligase APC/C-Cdh1 from the analysis of in vivo degrons

Molecular Biology of the Cell ◽

10.1091/mbc.e14-09-1342 ◽

2015 ◽

Vol 26 (5) ◽

pp. 843-858 ◽

Cited By ~ 11

Author(s):

Lea Arnold ◽

Sebastian Höckner ◽

Wolfgang Seufert

Keyword(s):

Nuclear Localization ◽

Ubiquitin Ligase ◽

Budding Yeast ◽

Cellular Mechanism ◽

Nuclear Localization Sequence ◽

Localization Sequence ◽

In Vivo Analysis ◽

Spatiotemporal Control

In vivo analysis in budding yeast identifies APC/C-Cdh1–specific minimal degrons carrying either a D or a KEN box and a nuclear localization sequence. APC/C-Cdh1 activity is restricted to the nucleus, maximal in the nucleoplasm, and absent from the cytoplasm, allowing for spatiotemporal control of Cdh1 substrate proteolysis.

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