A. N. Belozersky Institute of Physico-Chemical Biology and Department of Virology, Moscow State University, Moscow 119899, Russia1
Institute of Carcinogenesis, Cancer Research Center, Moscow 115478, Russia2
Institute of Plant Virology, Microbiology and Biosafety, Federal Biological Research Centre for Agriculture and Forestry, Messeweg 11/12, D-38104 Braunschweig, Germany3
Department of Biochemistry and Pharmacy, bo Akademi University, 20521 Turku, Finland4
Department of Biology, University of Turku, 20500 Turku, Finland5
Author for correspondence: Sergey Yu. Morozov. Fax +7 095 939 31 81. e-mail morozov{at}genebee.msu.su
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Abstract |
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Introduction |
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On the other hand, the MPs of TMV and alfalfa mosaic virus were shown also to interact with the endoplasmic reticulum (ER), thus displaying the properties of integral membrane proteins (Heinlein et al., 1998 ; Reichel & Beachy, 1998
; Huang & Zhang, 1999
, Reichel et al., 1999
; Beachy & Heinlein, 2000
). Recently, the sequence domain responsible for interaction of the TMV MP with membranes was identified (Brill et al., 2000
). This protein region was shown to be a portion of a hydrophobic core in a large family of plant virus MPs (Melcher, 2000
), suggesting that membrane interaction could be a general feature of plant virus MPs.
The first membrane-bound MPs to be identified were encoded by the triple gene block (TGB), a module of three MP genes conserved in a number of virus genera (Morozov et al., 1989 ; Rupasov et al., 1989
; Petty & Jackson, 1990
; Beck et al., 1991
; Gilmer et al., 1992
; Koenig et al., 1998
; Lawrence & Jackson, 2001a
). Small proteins encoded by the second and third TGB genes (TGBp2 and TGBp3) were originally predicted to have transmembrane sequence segments and to therefore represent a novel type of integral membrane MP (Morozov et al., 1987
, 1989
, 1990
; Skryabin et al., 1988
). In further studies, apart from TGB proteins, membrane-binding properties were demonstrated or predicted for the MPs of a number of virus groups. These included RNA viruses of the families Tombusviridae (genera Carmovirus, Necrovirus, Machlomovirus and Panicovirus) (Hacker et al., 1992
; Molnar et al., 1997
; Huang et al., 2000
) and Closteroviridae (Agranovsky et al., 1991
; Alzhanova et al., 2000
) and DNA viruses of the genera Mastrevirus (Mullineaux et al., 1988
; Boulton et al., 1993
) and Nanovirus (Burns et al., 1995
; Katul et al., 1997
).
Recently, the properties of the hydrophobic mastrevirus MP have been studied. Similar to the membrane-bound TMV MP, the MP of maize streak virus was able to move as a GFP fusion from transfected leaf cells to neighbouring cells (Kotlizky et al., 2000 ). Additionally, mastrevirus MP interacted with viral coat protein (CP) and directed the transport of CP and, presumably, encapsidated viral DNA to the cell periphery (Kotlizky et al., 2000
; Liu et al., 2001
). Importantly, specific features of the hydrophobic membrane-embedded segments of mastrevirus MPs are responsible for protein trafficking, which directs cell-to-cell translocation of the viral genome (Kotlizky et al., 2000
).
A recent study of poa semilatent hordeivirus (PSLV) membrane proteins TGBp2 and TGBp3 demonstrated that membrane association of hordeivirus MPs is required for directed translocation of MPs and, potentially, viral RNA to peripheral cell compartments. Using GFP-tagged proteins, it was shown that PSLV TGBp2 associated mostly with the ER, whereas TGBp3 was found in membrane bodies of different size located at the cell periphery along the plasma membrane. Coexpression of PSLV TGBp2 and TGBp3 demonstrated that TGBp3 was able to direct TGBp2 from the ER structures to the peripheral bodies (Solovyev et al., 2000 ). Taking into account the RNA-binding activity of the hordeivirus TGBp1, which is able to form virus-specific ribonucleoproteins (RNPs) (Brakke et al., 1988
; Donald et al., 1997
; Kalinina et al., 2001
), and the requirement of both TGBp2 and TGBp3 for specific direction of TGBp1 to plasmodesmata-associated sites (Erhardt et al., 1999
, 2000
; Lawrence & Jackson, 2001b
), it was proposed that the role of TGBp3 in viral transport could involve intracellular translocation of TGBp2 and viral RNPs to plasmodesmata (Solovyev et al., 2000
; Tamai & Meshi, 2001
). The TGBp3-directed subcellular sorting of TGBp2 seems to be sequence-nonspecific, as inferred from mutagenesis of both proteins and the observation that the potato virus X TGBp3 protein, having no sequence relation to PSLV TGBp3, was able to target PSLV TGBp2 to peripheral bodies (Solovyev et al., 2000
).
In this paper, we used dual-colour imaging with two fluorescent proteins, GFP and the red fluorescent protein DsRed, to analyse the TGBp3-directed targeting of TGBp2 and non-related membrane proteins in plant cells. Additionally, we demonstrate that intracellular co-transport of TGBp2 and TGBp3 can also occur in mammalian cells.
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Methods |
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Fluorescent microscopy.
Fluorescence was detected using a Zeiss Axioscope 20 fluorescence microscope. For GFP detection, excitation filter BP450490, beam splitter FT510 and emission filter HQ535/50x were used. For DsRed detection, excitation filter HQ565/30, beam splitter Q585LP and emission filter HQ620/60 were used. Confocal laser scanning microscopy was done on a Zeiss CLSM 410 system with Kr/Ar laser (488 nm) for GFP excitation and He/Ne laser (543 nm) for DsRed excitation. Emission from GFP and DsRed was detected using bandpass filters 510525 nm or 560580 nm, respectively. Projection of serial optical sections was performed using Carl Zeiss LSM software (version 3.98). Measurement of red vs green intensity in each pixel of the optical section was done using the program Comparator, kindly provided by I. V. Skulachev and K. V. Skulachev.
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Results |
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Coexpression of TGBp3 with GFP targeted to the ER lumen
To analyse the possible relation of the TGBp3-containing peripheral membrane bodies to the ER structures, we visualized ER using m-GFP5-ER, a GFP derivative containing the N-terminal signal sequence of Arabidopsis thaliana basic chitinase and C-terminal ER retention signal (Boevink et al., 1998 , 1999
). In epidermal cells of N. benthamiana leaves bombarded with pRT-m-GFP5-ER, the GFP fluorescence was confined to the ER (Fig. 1d
). In cobombardment experiments with pRT-m-GFP5-ER and pRT-18K, the typical ER structures remained unaffected; however, in addition to these structures peripheral bodies were observed which resembled those found in the cells expressing GFP-18K (Solovyev et al., 2000
; Fig. 1e
). Furthermore, after cobombardment of pRT-m-GFP5-ER and pRT-DsRed-18K, all the DsRed-18K-containing peripheral bodies also contained the ER-targeted GFP and seemed to be connected to the cortical ER network (Fig. 1f
h
). Also, in the cells showing lower overall red fluorescence, the size of the peripheral bodies was much smaller (Fig. 1f
h
) compared to the cells with higher levels of 18K expression (Fig. 1e
and data not shown). possibly because of lower expression levels of DsRed-18K, Presumably, the size of the peripheral bodies correlates with the amount of the TGBp3 protein in a particular cell.
Coexpression of TGBp3 with non-TGB membrane-associated proteins
To analyse whether TGBp3 was also able to influence localization of non-viral membrane-embedded proteins, we constructed two GFP derivatives carrying artificial hydrophobic tail sequences. Plasmid pRT-GFP-A expressed the modified GFP fused to a C-terminal pseudo-random sequence, GSLLVILLLIALLILVLLSK, with 18 residues potentially comprising a membrane anchoring signal (Sääf et al., 1998 ). Plasmid pRT-L-GFP-A carried another modification of the GFP gene which, in addition to the C-terminal anchor, encoded the N-terminal leader MKGSLLIVILVLILALVLLIES, which also includes an artificial 18 residue hydrophobic sequence. Particle bombardment of N. benthamiana leaves with pRT-GFP-A and pRT-L-GFP-A demonstrated that both GFP derivatives were localized to the ER structures, showing a fluorescence pattern similar to that of m-GFP5-ER (Fig. 1d
, i
). Cobombardment of pRT-DsRed-18K with pRT-GFP-A or pRT-L-GFP-A resulted in GFP localization to both typical ER elements and DsRed-18K-containing peripheral structures, phenotypically resembling coexpressed m-GFP5-ER and DsRed-18K (Fig. 1i
k
and data not shown).
To obtain further insight into the mechanism of TGBp3-directed protein targeting, we analysed the ability of DsRed-18K to influence subcellular localization of GFP-tagged membrane proteins encoded by two unrelated plant viruses, the DNA-containing faba bean necrotic yellows nanovirus (FBNYV) and the RNA-containing beet yellows closterovirus (BYV).
The putative movement protein encoded by genomic DNA component 4 (C4) of FBNYV is homologous to the banana bunchy top virus (BBTV) DNA4-encoded protein (Burns et al., 1995 ; Katul et al., 1997
), which was shown to localize to membranes at the cell periphery (Wanitchakorn et al., 2000
). Bombardment of N. benthamiana leaves with plasmid pRT-GFP-FBNYV4 revealed that the GFP-tagged FBNYV C4 protein was dispersed along the cell boundary, and no fluorescence was observed in the cell interior (Fig. 2a
, b
). This was similar to the reported localization of the BBTV DNA4-encoded protein in banana embryogenic cells (Wanitchakorn et al., 2000
). Cobombardment of pRT-GFP-FBNYV4 with pRT-DsRed-18K demonstrated the precise colocalization of red and green fluorescence in the peripheral bodies typical for DsRed-18K (Fig. 2d
f
), showing that the FBNYV protein was targeted by TGBp3 to these structures.
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Targeting of TGBp2 and TGBp3 in mammalian cells
To verify if the mechanism of protein subcellular co-sorting exploited by TGBp2/TGBp3 is universal for eukaryotes, we expressed the TGBp2 and TGBp3 proteins of PSLV in mammalian cells. The GFP-15K and DsRed-18K genes were subcloned under control of the cytomegalovirus (CMV) IE promoter, and the resulting plasmids, pIE-GFP-15K and pIE-DsRed-18K, were used for transfection of baby hamster kidney (BHK-21) cells and 293T cells (a human embryonic kidney line). In both cell lines, the localization of PSLV proteins was similar: therefore only the data obtained with BHK-21 cells are presented in this paper.
In BHK-21 cells transfected with pIE-GFP-15K, distribution of the GFP fluorescence suggested localization of GFP-15K to the ER (Fig. 3ad
). As GFP-15K localized to the ER in plant cells (Solovyev et al., 2000
), this finding indicates that there is a common mechanism responsible for such protein targeting in plant and animal cells. DsRed-18K in transfected BHK cells was localized to inclusion bodies of different sizes (Fig. 3e
h
). Larger bodies could not be assigned to an obvious cell membrane compartment. However, confocal laser scanning microscopy of DsRed-18K-expressing cells demonstrated that in single optical sections crossing a cell near the surface the numerous small bodies were located mostly in the peripheral cytoplasm or plasma membrane (Fig. 3g
). Similar bodies were observed in BHK-21 cells expressing GFP-18K (data not shown), demonstrating that such localization was not due to the particular fluorescent tag but instead reflects specific targeting of TGBp3 in mammalian cells.
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Coexpression of TGBp3 with caveolin-1 in mammalian cells
In BHK-21 cells transiently transfected with pIE-DsRed-18K, fluorescence at the cell border was mostly localized to numerous tiny inclusions (Fig. 3g), which is similar to the localization pattern previously described for GFP-tagged caveolin-1 (Volonté et al., 1999
), the main structural protein of caveolae, omega-shaped invaginations of the plasma membrane (Okamoto et al., 1998
). To determine if TGBp3 moves to the same destination sites as caveolin-1 and has an effect on localization of a membrane protein specific for mammalian cells, we used vector pIE-GFP-CAV1 expressing human caveolin-1 (CAV1) tagged with GFP. When pIE-GFP-CAV1 was transfected into BHK-21 cells alone or together with pIE-DsRed-18K, GFP distribution was typical for GFP-tagged caveolin-1 (Fig. 3i
l
) (Volonté et al., 1999
); however, colocalization of the two proteins was only partial (Fig. 3q
t
). Computer-assisted analysis of a number of cell images scoring the percentage of pixels with both red and green signals showed that red/green colocalization in DsRed-18K/GFP-15K coexpression was greater than 80%, whereas in the case of DsRed-18K/GFP-CAV1 this parameter was less than 45%, confirming the lower cotargeting efficiency of 18K and CAV1. These data suggest that DsRed-18K and GFP-CAV1 have little (if any) effect on the localization of each other in mammalian cells, although CAV1 is also known to cotarget a number of other proteins including caveolin-2 (Okamoto et al., 1998
; Mora et al., 1999
; Parolini et al., 1999
; Razani et al., 1999
).
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Discussion |
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In an attempt to resolve the nature of the peripheral bodies, which represent membrane structures (Solovyev et al., 2000 ), DsRed-18K was coexpressed with m-GFP5-ER, a GFP derivative localized to the ER lumen (Boevink et al., 1998
, 1999
). The peripheral bodies where DsRed-18K was localized contained m-GFP5-ER and seemed to be connected to the cortical ER network (Fig. 1f
h
), suggesting that these bodies could originate from a subdomain of the cortical ER. Moreover, such bodies were never observed in cells expressing only m-GFP5-ER (Fig. 1d
), and their size correlated with the expression level of 18K (Fig. 1e
and data not shown). Thus it can be suggested that TGBp3 causes modification and proliferation of this ER subdomain. Formation of cortical bodies originating from ER membranes and containing virus MP has been observed in the epidermal cells of N. benthamiana infected with TMV, and simultaneous partial disruption of the ER network was found to be induced by the TMV MP (Reichel & Beachy, 1998
). Accordingly, fewer ER tubules can be observed in some cells expressing TGBp3 (Fig. 1d
, e
). At early and middle stages of TMV infection, similar modifications of the ER were attributed to the intermediate steps of intracellular movement of MP and virus RNP toward plasmodesmata (Reichel & Beachy, 1998
).
The hypothesis that TGBp3 is localized to a membrane compartment which originated from the cortical ER implies that either trafficking of TGBp3 from the sites of synthesis to the peripheral bodies takes place without exiting the ER network or TGBp3 is translocated to the destination sites in ER-derived specific containers that do not fuse to cis-Golgi compartments from which rapid retrograde transport of m-GFP5-ER occurs (for review see Hawes et al., 1999 ; Hadlington & Denecke, 2000
). Although the general path that proteins travel to their destination sites is thought to involve the Golgi apparatus, a Golgi-independent pathway for protein transport from the ER has been suggested for plant cells (for review see Hawes et al., 1999
; Vitale & Raikhel, 1999
). For example, transport of some soluble and membrane proteins to storage vacuoles may occur without their entering the Golgi in large ER-derived vesicles (Jiang & Rogers, 1998
; Chrispeels & Herman, 2000
; Frigerio et al., 2001
; Mitsuhashi et al., 2001
). Presumably, TGBp3 translocation to the cell periphery and TGBp2 cotargeting also involve this mechanism.
In further experiments we analysed the ability of TGBp3 to affect the localization of artificial integral membrane proteins. Two GFP derivatives were constructed carrying either C-terminal, or both N- and C-terminal, artificial hydrophobic sequences that would target the proteins to membranes. Indeed, when expressed alone, both GFP derivatives were associated with the ER membranes (Fig. 1i and data not shown), in agreement with observations that ER retention of proteins in plant cells can be directed by transmembrane amino acid stretches (Sato et al., 1999
). For example, in the absence of any other hydrophilic signals, static retention of protein in the ER can be caused by the presence of rather short hydrophobic domains of 1821 residues which, when lengthened, result in leakage from the organelle and trafficking to the vacuole or plasma membrane (Honsho et al., 1998
; Pedrazzini et al., 2000
; Sato et al., 2001
). After coexpression with DsRed-18K, both membrane-embedded GFP derivatives were detected in the ER network and TGBp3-containing peripheral bodies, showing a localization phenotypically similar to that of m-GFP5-ER (Fig. 1f
k
and data not shown). These observations demonstrated that anchoring to the ER membranes per se is not sufficient for TGBp3-induced redirection of a protein to peripheral bodies.
We have previously demonstrated that TGBp3-directed targeting of TGBp2 could occur in a sequence-independent manner (Solovyev et al., 2000 ). To verify if the natural non-TGB membrane proteins are able to be targeted to peripheral bodies by PSLV TGBp3, we analysed two viral integral membrane MPs, the 6 kDa (6K) protein encoded by BYV (Agranovsky et al., 1991
) and p4, the 13 kDa protein encoded by FBNYV DNA component 4 (Katul et al., 1997
). FBNYV p4, when expressed as a GFP fusion in N. benthamiana cells, was localized to the cell cortex close to the plasma membrane (Fig. 2a
b
), similar to the reported localization of a homologous protein, the gene product of BBTV nanovirus DNA4, in isolated banana cells (Wanitchakorn et al., 2000
). The BYV GFP-6K protein was associated with the ER network (Fig. 2c
). Interestingly, the closterovirus membrane MPs exhibit a sequence conservation only in their putative transmembrane domain, but not in the hydrophilic protein regions (Agranovsky, 1996
), suggesting that signal(s) responsible for the retention of the BYV 6K protein in ER could be localized in the hydrophobic stretch of amino acids. Indeed, a key role of transmembrane domains in maintaining ER residency by static retention or dynamic retrieval from the Golgi has been shown (Pedrazzini et al., 1996
, 2000
; Rayner & Pelham, 1997
; Letourneur & Cosson, 1998
; Sato et al., 1999
, 2001
).
Although BYV 6K and FBNYV p4 have no sequence similarity and differ significantly in their subcellular localization, both proteins were targeted by the PSLV TGBp3 to peripheral bodies (Fig. 2df
and Fig. 2j
l
). This finding confirms that TGBp3-directed targeting of membrane proteins can occur in a sequence-independent manner and suggests that the TGBp3-induced relocation of the membrane proteins does not occur from their normal destination compartments, but directly from the sites of their synthesis in rough ER. Generally, specific features of membrane proteins, such as the nature of their sites of primary localization and normal targeting pathway, could contribute to the ability to be targeted by TGBp3.
Since the cellular protein sorting machinery is thought to be conserved in yeast, plants and animals (Hawes et al., 1999 ; Sato et al., 1999
; Andreeva et al., 2000
; Batoko et al., 2000
; Nebenführ & Staehelin, 2001
), it is not surprising that sorting signals of plant proteins are also functional in other systems (Altschuler et al., 1993
; Sato et al., 1999
; Kikkert et al., 2001
). In this paper we demonstrate that the TGBp3-directed relocation of TGBp2 occurs in mammalian cells (Fig. 3
), indicating that the cotargeting mechanism is not dependent on components specific for plant or animal cells. Since the animal plasma membrane protein caveolin-1 colocalizes poorly with TGBp3 after coexpression, it can be concluded that TGBp3- and CAV1-induced protein codirection in mammalian cells is affected by specific features of targeted membrane proteins (see also Mora et al., 1999
; Parolini et al., 1999
; Razani et al., 1999
). Importantly, in plant cells TGBp3 is targeted to the membrane structures closely associated with plasmodesmata (Solovyev et al., 2000
, and our unpublished data), which are apparently absent from mammalian cells. Therefore, although the TGBp3 destination sites in plant and mammalian cells may be different, the basic nature of protein trafficking signals and the cotargeting mechanism of the TGB proteins are universal.
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Acknowledgments |
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References |
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Received 30 July 2001;
accepted 16 November 2001.