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Targeting of Plant Rangap to the Nuclear Envelope Annkatrin Rose, Shalaka S

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Targeting of Plant Rangap to the Nuclear Envelope Annkatrin Rose, Shalaka S Targeting of Plant RanGAP to the Nuclear Envelope Annkatrin Rose, Shalaka S. Patel, Iris Meier Plant Biotechnology Center, Ohio State University, Columbus, OH 43210 Plant RanGAPs contain a unique N-terminal domain shared with the plant protein MAF1. Identification of an interaction partner of the WPP Introduction domain. The small GTPase Ran is one of the key players in A WPP domain Leucine-rich repeat Acidic SUMO attachment Figure 1: (A) Schematic comparison of the domain structure of vertebrate, yeast, and plant RanGAPs and MAF1 nucleocytoplasmic transport. The directionality of this HsRanGAP1 1 587 The WPP domain-binding protein FLIP2 is a novel coiled-coil (HsRanGAP1, human; ScRanGAP1, S. cerevisiae ; AtRanGAP1 transport is determined by a gradient of RanGTP and protein. ScRna1p 1 386 and AtMAF1, Arabidopsis). (B) Sequence alignment of RanGDP across the nuclear envelope. This gradient coiled-coil domain AtRanGAP1 1 535 Arabidopsis MAF and MAF-like sequences with the N-terminal is formed by the spatial separation of RanGAP (Ran domains of the two Arabidopsis RanGAPs. Blue shading AtMAF1 1 155 GTPase activating protein) outside and a nucleotide indicates amino acid identity, yellow shading similarity. The LeFLIP2 1 834 Coiled-Coil Region 29% 81% highly conserved WPP motif is boxed in red. Transmembrane exchange factor inside the nucleus. In mammalian AtFLIP2 1 829 1 M --------------------------------------------D B AtRanGAP1 19% 57% Region cells, the C-terminal domain of RanGAP is 1 M --------------------------------------------A AtRanGAP2 Highly Conserved 1 M AETETESITTSSPPPISETENSTTLP-----------------TT AtMAF1 RanGAPs from all three kingdoms contain a core leucine-rich OsFLIP2 1 747 associated with the outer surface of the nuclear pore 1 M AET-AETINTTISSPPPESESSTTISAMTDPTSQEAASKDTDLTK AtMAF2 Region 1 M AET-ADTINTTVSTPQPQLESRSD-----ETSCLQKHRSDA--TS AtMLP repeat (red) and acidic domain (yellow) necessary for the complex. This mammalian nuclear envelope- 3 HSAKTTQNR VL S VK M WPP S KS TR LM LV E R MTKN IT T P SI F S R K YG L AtRanGAP1 GTPase activating function. In mammalians, the SUMOylated Figure 5: FLIP2 (filament-like protein 2) was identified from tomato in a two- targeting domain is not present in yeast and plant 3 D ILDS-RPH A F S IKL WPP S LP TR KA LI E R ITNNF S S K T I F T E K YG S AtRanGAP2 30 E TEKNPNPVT I S LRI WPP T QK TR DA VI N R LIET L ST E SI L S K RF G S AtMAF1 C-terminal domain (green) is required for attachment to the hybrid screen with MAF1 and subsequently shown to bind to AtRanGAP1. The RanGAP proteins, therefore suggesting a different 46 E AESEKKPG GI S LRI WPP T QK TR DA VL N R LIET L ST E SI L S K R YG T AtMAF2 domain structure of the protein is depicted with the predicted coiled-coil 39 E VTKEEKSG GI LFS V WPP CQK S R DY VV NSMIKT L ST D SI L S Y K YG T AtMLP nuclear pore protein NUP358. Yeast RanGAP (ScRna1p) mechanism or no targeting of RanGAP to the nuclear regions of the tomato protein (LeFLIP2) and its Arabidopsis and rice homologs 49 L SV EE A EQD AK R IE D L A F AT A NKHFQNEP D G DG TS AV H V Y A KE S SK appears to be cytoplasmic. Plant RanGAP contains a N- AtRanGAP1 (AtFLIP2 and OsFLIP2, respectively) below. Percentages in red indicate envelope in those organisms. 48 L TK D Q A T EN AK R IE D I A F ST A NQQFEREP D G DG G S AV Q L Y A KE C SK AtRanGAP2 76 L ES EE A S SV AK S IE D E A Y AI A SATVF--G D D DG I E IL K A Y S KE I SK AtMAF1 terminal domain shared with the nuclear envelope protein amino acid identities. 92 L KS DD A T TV AK L IE E E A Y GV A SNAVS--S D D DG I K IL E L Y S KE I SK AtMAF2 85 I KP EE A S AV AK S IE E K A Y DI A SRFVS--S D -- G I KN L E V Y GI E T S E AtMLP MAF1 (cyan), indicating a possible role of this domain in 95 L ML D V I K --------RGPQEESE V -EV----------SKDGDVFF… AtRanGAP1 targeting plant RanGAP to the nuclear rim. Due to the The coiled-coil domain of LeFLIP2 specifically interacts with 94 LI LE V L K --------KGPVAKVA A REL----------ISEDSVSP… AtRanGAP2 Plant RanGAP and MAF1 are targeted to 120 R MLE S V K AKSN--VASPPPKDGD G IESAVD-------SKIDSSEA AtMAF1 presence of a conserved WPP sequence motif (red box), this the WPP domain of AtRanGAP1. 136 R MLE S V K ARSNASVGNGSVEDANTDASEVSKDDAGPASEEEKSEA AtMAF2 the nuclear envelope in plant cells. 127 R M I E S A EVRFKANGSMELLLNQT I KMMQLLI AtMLP domain is referred to as the WPP domain. LeFLIP2 LeFLIP2 ∆∆∆C LeFLIP2 ∆∆∆N A LeMAF1 + n.d. + AtMAF1 + n.d. + The WPP domain - a novel nuclear envelope targeting domain in plants. AtMAF2 + n.d. + AtRanGAP1 + - + AtRanGAP1 ∆∆∆N - - - The WPP domain is necessary and sufficient for nuclear- 40 41 42 aa The conserved WPP motif is required W P P AtRanGAP1 ∆∆∆C - + wildtype + envelope targeting of Arabidopsis RanGAP1. TGG CCA CCG for nuclear-envelope targeting of * AtRanGAP1 ∆∆∆Cmut - n.d. - A A P Arabidopsis RanGAP1. 10 µµµm mutant GCG GCA CCG A AtRanGAP1-GFP Figure 6: Interaction specificities of MAF and RanGAP with FLIP2. Proteins are depicted as bar diagrams. Color code of the AtRanGAP1 domains is as in B * Fig. 1 (A), color code for the LeFLIP2 domains as in Fig. 5. +, interaction; -, no WPP LRR Acidic GFP A AtRanGAP1mut-GFP interaction; n.d., not determined. The interactions found in two-hybrid assays were verified by in vitro binding experiments. LeFLIP2 is a novel coiled-coil protein with unknown function that * interacts with all tested WPP-domain proteins and binds to the WPP B AtRanGAP1 ∆∆∆N-GFP domain of AtRanGAP1 through its coiled-coil domain. Mutation of WP to B AtRanGAP1 ∆∆∆Cmut-GFP AA in the conserved WPP sequence motif disrupts this interaction. C GFP Figure 2: GFP (green fluorescent protein) Conclusions and Outlook fluorescence of tobacco BY-2 cells transiently C AtRanGAP1 ∆∆∆C-GFP We have found fundamental differences in plant and animal RanGAP targeting expressing Arabidopsis RanGAP1-GFP (A) and to the nuclear envelope, suggesting that different mechanisms have evolved for tomato MAF1-GFP-MAF1 (B) fusion proteins. The Figure 4: GFP fluorescence of BY-2 cells transiently expressing the spatial organization of Ran signaling in plants and animals. The consistency MAF1 (MFP1-associated factor 1) sandwich construct constructs with a mutation of the WPP motif. The wildtype sequence between nuclear-envelope targeting and binding of plant RanGAP to FLIP2 was designed to prevent passive diffusion into the and introduced sequence changes are indicated above the bar diagram indicates that interaction with FLIP2 might be involved in anchoring plant nucleus. See also figure on top next to title: Top left Figure 3: GFP fluorescence of BY-2 cells transiently expressing of AtRanGAP1mut-GFP (A). The asterisk indicates the position of the cell: AtRanGAP1-GFP, bottom right cell: LeMAF1- identical mutation in AtRanGAP1 ∆Cmut-GFP (B). The fluorescence of RanGAP to the nuclear rim. This hypothesis and six additionally identified AtRanGAP1-GFP (A), AtRanGAP ∆N-GFP (B), and AtRanGAP ∆C-GFP GFP-LeMAF1. SYTO 82 orange was used to free GFP is shown as a control (C). Bars in all microscopy pictures are RanGAP-binding proteins from Arabidopsis are currently under investigation. counterstain for DNA (red), labeling the nucleus and (C). 10 µm. nucleolus, as well as mitochondria and plastids in the cytoplasm. Deletion of the WPP domain (B) leads to loss of fusion protein at The mutation of the conserved WPP motif to AAP in AtRanGAP1 A concentration of GFP fusion protein around the Literature the nuclear rim, whereas the WPP domain alone (C) is sufficient to abolishes the targeting of the GFP fusion protein to the nuclear Meier, I. (2000): A novel link between Ran signal transduction and nuclear envelope proteins in nuclear rim can be observed for both Arabidopsis target GFP to the nuclear envelope. rim. plants. Plant Physiol. 124 , 1507-1510. RanGAP1 and tomato MAF1. Rose, A. and Meier, I. (2001): A domain unique to plant RanGAP is responsible for its targeting to the plant nuclear rim. Proc. Natl. Acad. Sci. U.S.A. 98 , 15377-15382..
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