1 A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119899, Russia
2 Institute of Plant Virology, Microbiology and Biosafety, Federal Biological Research Centre for Agriculture and Forestry, Messeweg 11/12, D-38104 Braunschweig, Germany
3 Natural Sciences Center of A. M. Prokhorov, General Physics Institute, Russian Academy of Sciences, Bld L-2, 38 Vavilov Str., Moscow 119991, Russia
4 Department of Plant Biology and Forest Genetics, Swedish University of Agricultural Sciences (SLU), PO Box 7080, S-750 07 Uppsala, Sweden
Correspondence
S. Yu. Morozov
morozov{at}genebee.msu.su
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ABSTRACT |
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INTRODUCTION |
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In general, cell-to-cell movement through plasmodesmata and long-distance transport of viral/viroid RNAs and some endogenous RNAs in plants is believed to occur in the form of specific ribonucleoprotein complexes (RNPs) (Carrington et al., 1996; Tzfira et al., 2000
; Gómez & Pallás, 2004
; Lucas & Lee, 2004
; Oparka, 2004
; Waigmann et al., 2004
; Heinlein & Epel, 2004
). In potexviruses, transport-competent RNPs contain TGBp1, the largest TGB protein, and the viral CP (Lough et al., 1998
, 2000
). PVX TGBp1 is able to modify plasmodesmata and move between cells (Angell et al., 1996
; Lough et al., 1998
, 2000
; Yang et al., 2000
; Krishnamurthy et al., 2002
; Howard et al., 2004
; Verchot-Lubicz, 2005
). Various evidence suggests that potexviral transport-competent RNPs could represent virions modified by TGBp1 (Santa Cruz et al., 1998a
; Atabekov et al., 2000
). TGBp1 associates in vitro with one end of filamentous virions, inducing their structural remodelling (Atabekov et al., 2000
; Rodionova et al., 2003
). Accordingly, TGBp1 contains an NTPase/helicase domain (Koonin & Dolja, 1993
; Morozov & Solovyev, 2003
) and demonstrates NTPase, RNA helicase and RNA-binding activities in vitro (Kalinina et al., 1996
, 2001
, 2002
; Lough et al., 1998
; Morozov et al., 1999
; Wung et al., 1999
; Hsu et al., 2004
).
In rod-shaped viruses, TGBp2 and TGBp3 move to plasmodesmata and act in concert to transport TGBp1 to and through plasmodesmata (Erhardt et al., 2000; Lawrence & Jackson, 2001
; Gorshkova et al., 2003
; Zamyatnin et al., 2004
; Haupt et al., 2005
), suggesting a role in intracellular delivery of transport-competent RNPs carrying viral genomes. Recent studies have indicated that TGBp2 and TGBp3 may have additional movement-related functions (Morozov & Solovyev, 2003
). In particular, potexvirus TGBp2, the most conserved TGB protein (Koenig et al., 2004
), affects the protein-trafficking capacity of plasmodesmata and facilitates movement of green fluorescent protein (GFP) between adjacent epidermal cells (Tamai & Meshi, 2001
; Lucas & Lee, 2004
). This activity may be a result of association with TIP, a host protein regulator of
-1,3-glucanase, which is the key enzyme of callose turnover (Fridborg et al., 2003
). However, recent evidence suggests that the TGBp2 interaction with the plasmodesmata gating mechanism can be different in various plant hosts (Krishnamurthy et al., 2002
; Mitra et al., 2003
; Verchot-Lubicz, 2005
).
PVX TGBp2 and TGBp3 exhibit properties of integral membrane proteins (Morozov et al., 1987, 1990
; Krishnamurthy et al., 2003
). Similar properties have been revealed for the TGBp2 and TGBp3 of rod-shaped viruses (Morozov & Solovyev, 2003
). In these viruses, TGBp3, which has two transmembrane domains, is localized to peripheral endoplasmic reticulum (ER)-related membrane bodies adjacent to the plasma membrane and directs TGBp2 and TGBp1 (in the presence of TGBp2) to peripheral bodies (Solovyev et al., 2000
; Zamyatnin et al., 2002
; Cowan et al., 2002
; Gorshkova et al., 2003
; Haupt et al., 2005
). The functional relevance of the TGBp3-containing peripheral bodies has been confirmed recently by observations showing that mutants of Potato mop-top virus (PMTV, genus Pomovirus) TGBp3 that are unable to form peripheral bodies are also deficient in directing TGBp1 to and through plasmodesmata (Zamyatnin et al., 2004
). However, formation of peripheral bodies is not sufficient for cell-to-cell movement. For example, heterologous TGBp2 and TGBp3 directed PMTV TGBp1 to peripheral bodies; however, TGBp1 was unable to be transported through plasmodesmata (Zamyatnin et al., 2004
). Additionally, cell-to-cell movement of Beet necrotic yellow vein virus was severely inhibited by overexpression of heterologous TGBp3, despite the presence of authentic wild-type TGBp3 (Lauber et al., 2005
).
Molecular signals responsible for TGBp3 trafficking to peripheral bodies are yet to be elucidated. Sequence analyses of TGBp2 and TGBp3 in both filamentous and rod-shaped viruses (Morozov & Solovyev, 2003) failed to identify canonical signals of subcellular sorting (reviewed by van Vliet et al., 2003
). On the other hand, in rod-shaped Poa semilatent virus (PSLV, genus Hordeivirus), signals responsible for TGBp3 localization to peripheral bodies have been mapped to the central hydrophilic protein region conserved among TGBp3 of rod-shaped viruses and the second C-terminal transmembrane domain (Solovyev et al., 2000
). Both these structural elements are absent from PVX TGBp3. Indeed, potexviral TGBp3 proteins contain a single N-terminal transmembrane segment and a characteristic conserved hydrophilic sequence, which is not similar to that of rod-shaped viruses (Morozov et al., 1991
; Morozov & Solovyev, 2003
).
In this study, the influence of C-terminal deletions in PVX TGBp3 on virus cell-to-cell movement was examined and protein functionality and subcellular distribution were analysed in transient expression assays. Our data confirm that PVX TGBp3 acts intracellularly and does not move from infected cells to neighbouring ones. It has also been demonstrated that fusion of GFP directly to the N-terminal hydrophobic region abrogates TGBp3 activities in intracellular trafficking and complementation tests. However, TGBp3 C-terminal truncation mutants retain the ability of wild-type protein to complement (at least partially) PVX cell-to-cell movement and to direct the intracellular movement of GFP-TGBp3. Interestingly, intracellular transport of TGBp3 from sites of its synthesis in the rough ER to the cell periphery involves a non-conventional protein trafficking pathway.
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METHODS |
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To obtain ST-YFP, a region of rat -2,6-sialyl transferase gene encoding the transmembrane domain and short cytoplasmic tail (52 aa) was amplified by RT-PCR of rat liver RNA (minus-sense primer 5'-GACCCCATGGCCACTTTCTCCTGGCTCTTGGGC-3' and plus-sense primer 5'-CGCCCTCGAGATGATTCATACCAACTTGAAGAAAAAGTTCAGCC-3') and fused to the 5' terminus of the yellow fluorescent protein (YFP) gene (Clontech Laboratories).
To obtain pRT-GFP-8K, the PVX TGBp3 gene was amplified with specific primers 5'-GGGGGATCCATGGAAGTAAATACATATCTC-3' and 5'-GGGTCTAGATCAATGGAAACTTAACCGTTC-3'. The resulting product was digested with BamHI and XbaI and cloned into similarly digested pRT-GFP-15K (Solovyev et al., 2000).
To construct pRT-12K/8K, pRT-12K/8K24, pRT-12K/8K
28 and pRT-12K/8K
39, the corresponding 12K/8K gene sequences were amplified with specific primers. The forward primer 5'-CTCGAGATGTCCGCGCAGGG-3', common for these constructs, carried a XhoI site upstream of the 12K gene sequence. The reverse primers, 5'-GCGCGGATCCTCAATGGAAACTTAACCG-3' for pRT-12K/8K, 5'-GGATCCTCAGCAAGCCAACACTGTGA-3' for pRT-12K/8K
24, 5'-GGGTCTAGATCAATGGAAACTTAACCGTTC-3' for pRT-12K/8K
28 and 5'-GGATCCTCAAGGTTCAGTCCTCACTAAGG-3' for pRT-12K/8K
39, carried a BamHI site followed (for deletion mutants) by a stop codon downstream of the truncated 8K gene sequence. After cloning into pGEM-T (Promega), the amplified sequence was excised with XhoI and BamHI and inserted into similarly digested pRT103 (Töpfer et al., 1988
).
To replace the initiator codon of the PVX TGBp3 gene with ACG, a subcloned TGBp2/TGBp3-containing region of the PVX genome was amplified as two products, one obtained with an upstream primer and the specific primer 5'-GTATTTCGAACCGTAGATCAGCAAAGTCAGTAGC-3' and another with a downstream primer and the specific primer 5'-ATGGTTCGAAATACATATCTCAACGCAATC-3'. The specific primers introduced the initiator codon mutation and contained the AsuII restriction site. After digestion with ApaIAsuII and AsuIINheI, both amplification products were cloned into pPVX.GFP to give pPVX.GFP.AUG8K.
pPVX.15S (kindly provided by D. C. Baulcombe, Sainsbury Laboratory, Colney, UK) represented a full-length cDNA copy of the PVX genome containing a TGBp2 gene mutation (an insertion of four bases, AGCT, after nt 5251 of the PVX genome) that formed a translation terminator in the TGBp2 gene sequence. To obtain pPVX.GFP.15S, the region encoding TGBp2 and TGBp3 was excised from pPVX.15S and cloned into pPVX.GFP (Fedorkin et al., 2000) to replace the wild-type sequence.
Plant material.
Nicotiana benthamiana plants were grown in a greenhouse in soil (25 °C, 1012 h daylight). The largest fully expanded leaves were detached from 6-week-old plants. Such N. benthamiana leaves have been previously reported to represent transition (sink to source) leaves (Crawford & Zambryski, 2001). The level of GFP diffusion depends significantly on sink or source leaf status (Crawford & Zambryski, 2001
). However, using leaves of the same age for comparative analysis allows us to avoid precise detection of this status in the cases with considerable differences in the percentage of foci showing protein egress from initially bombarded cells.
Particle bombardment and fluorescent microscopy.
Particle bombardment of detached N. benthamiana leaves was performed using the flying-disk method with a high-pressure helium-based PDS-1000 apparatus (Bio-Rad) as described by Morozov et al. (1997). GFP fluorescence was detected with a Zeiss Axioscope 20 fluorescence microscope (excitation filter BP 450490; chromatic beam splitter FT 510 and band-pass filter HQ 535/50x) or Leica TCS SP2 confocal laser scanning imaging system with excitation light of 488 nm produced by an argon laser.
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RESULTS |
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In co-bombardment with PVX.GFP.AUG8K, the three C-terminally truncated TGBp3 mutants demonstrated a similar level of movement complementation. Compared with the foci produced by the movement-deficient PVX.GFP.
AUG8K, the number of foci consisting of one cell was decreased in co-expression experiments from 83·0 to 23·428·3 %, whereas the percentage of foci with three or more infected cells increased from 12·0 to 46·957·1 % (Fig. 2
). Accordingly, complementation resulted in an increase in the mean focus size (Fig. 2
). These data show that even complete deletion of the conserved hydrophilic region of PVX TGBp3 (mutant 8K
39) did not block the ability of mutant to complement cell-to-cell transport of PVX.GFP.
AUG8K. However, the efficiency of complementation was obviously lower in comparison with the wild-type TGBp3 gene (Fig. 2
). These data confirm earlier findings showing that the 8K gene mutant expressing only the N-terminal protein half is still capable of supporting a restricted virus cell-to-cell movement (Santa Cruz et al., 1998b
).
Subcellular localization and lack of functional competence of PVX TGBp3 fused to GFP
For subcellular localization studies, the GFP gene was fused to the 5' end of the PVX TGBp3 gene as described by Krishnamurthy et al. (2002) and placed under the control of the CaMV 35S RNA promoter producing the expression vector pRT-GFP-8K. Fluorescent microscopy of N. benthamiana leaves bombarded with pRT-GFP-8K revealed association of the fusion protein with ER tubules (Fig. 3
a), as previously reported by Krishnamurthy et al. (2003)
.
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Previously, wild-type PVX TGBp3 has been shown to direct GFP-fused TGBp2 to peripheral bodies (Solovyev et al., 2000), whereas bombardment of N. benthamiana leaves with pRT-GFP-12K alone revealed that most of the individually expressed TGBp2 of PVX were associated with numerous vesicles of an unknown nature (Fig. 3e
). This type of TGBp2 distribution resembles the behaviour of GFP-12K in Nicotiana tabacum cells (Mitra et al., 2003
). However, co-bombardment of pRT-GFP-12K with pRT-8K resulted in redirection of GFP-fused TGBp2 to peripheral bodies (Fig. 3f
) (Solovyev et al., 2000
). To test whether GFP-fused PVX TGBp3, like the wild-type protein, is able to influence the subcellular localization of PVX TGBp2 (Solovyev et al., 2000
), leaves were co-bombarded with pRT-GFP-12K and pRT-GFP-8K. In transfected cells, GFP fluorescence was associated with the ER network (Fig. 3g
), whereas peripheral bodies typical of co-expression of GFP-fused TGBp2 and non-fused TGBp3 (Fig. 3d
) were never observed. These data demonstrate that the fused GFP molecule hindered the ability of TGBp3 to target TGBp2 to peripheral bodies.
The functional competence of GFP-8K was also tested in co-bombardment of pPVX.GFP.AUG8K and pRT-GFP-8K. In this experiment, both the mean size of infection foci and the percentage of foci consisting of one cell were similar to those in pPVX.GFP.
AUG8K-bombarded control leaves (Fig. 2
). In contrast, the non-fused 8K efficiently complemented cell-to-cell movement of pPVX.GFP.
AUG8K (Fig. 2
). These data demonstrate that the GFP fusion of PVX TGBp3 is dysfunctional.
PVX TGBp3-containing peripheral bodies are ER-derived membrane structures
Since TGBp3 expression caused relocation of GFP-fused TGBp3 from the ER network, one could propose that TGBp3 directed ER disruption and its reorganization into condensed peripheral bodies. To verify this hypothesis, N. benthamiana leaves were co-bombarded with three expression vectors, pRT-GFP-8K, pRT-8K and pRT-ER-YFP. The last plasmid carried the gene of ER-targeted YFP (Zamyatnin et al., 2004). In bombarded cells, as expected, GFP fluorescence was detected in peripheral bodies (Fig. 3j
). ER-YFP was found both in the typical ER structures and the peripheral bodies (Fig. 3k and l
). Thus, expression of TGBp3 did not disrupt the ER. Importantly, the presence of the ER marker in the TGBp3-containing peripheral bodies demonstrated that they represent membrane structures of an ER origin.
Subcellular localization of the C-terminally truncated mutants
When a mixture of vectors pRT-GFP-8K and pRT-12K/8K was used for bombardment of N. benthamiana leaves, GFP fluorescence was associated with the peripheral bodies as in the case of co-bombarded vectors pRT-GFP-8K and pRT-8K (Fig. 3d; data not shown). Thus, even a suppressed level of TGBp3 expression resulted in targeting of GFP-TGBp3 to peripheral bodies.
The expression vectors pRT-12K/8K24, pRT-12K/8K
28 and pRT-12K/8K
39 (Fig. 1
) were individually co-bombarded with pRT-GFP-8K. For all mutants, GFP fluorescence observed 2024 h after co-bombardment was associated with the peripheral bodies, which is similar to the situation found in co-bombardment of pRT-GFP-8K with pRT-12K/8K (Fig. 3h and i
; data not shown). Thus, even complete deletion of the conserved hydrophilic region of PVX TGBp3 (mutant 12K/8K
39) did not block the ability of the protein to reach the peripheral sites. These data suggest that signals responsible for intracellular protein trafficking to the peripheral compartments are located in the N-terminal hydrophobic sequence of PVX TGBp3.
TGBp3 intracellular transport to peripheral bodies is COPII-independent
In yeasts, animals and plants, the transport of membrane proteins from the ER to destination cell compartments involves, as a rule, COPII-coated vesicles (Nebenführ, 2002; Barlowe, 2003
; Bonifacino & Lippincott-Schwartz, 2003
; van Vliet et al., 2003
). Formation and budding of the COPII transport vesicles on the ER membranes requires a small GTPase, Sar1 (Andreeva et al., 2000
; Pasqualato et al., 2002
; Barlowe, 2003
). To determine whether the transport of TGBp3 from the ER to peripheral membrane bodies is COPII-dependent, Sar1[T39N], the previously described dominant negative mutant of Sar1 that prevents COPII budding complex formation (Andreeva et al., 2000
), was constructed.
To verify whether Sar1[T39N] had the expected effect on the COPII-dependent vesicular transport in our experimental system, a marker protein (ST-YFP) was used that represented YFP fused to the N-terminal signal anchor sequence of a rat sialyl transferase as described by Saint-Jore et al. (2002). This fusion has been previously shown to localize to Golgi stacks of plant cells and to be transported from ER to Golgi via the COPII pathway (Boevink et al., 1998
; Saint-Jore et al., 2002
; Brandizzi et al., 2004
). When expressed in N. benthamiana epidermal cells, ST-YFP was indeed found in Golgi stacks (Fig. 4
a). Co-expression of ST-YFP with Sar1[T39N] resulted, as described previously (Andreeva et al., 2000
; Nebenführ, 2002
; Ritzenthaler et al., 2002
), in localization of YFP fluorescence in ER elements (Fig. 4b
) or an ER-like network partially converted to a system of lamellar cisterns punctured by irregularly sized holes (Fig. 4c
). Presumably, these two non-mutually-exclusive phenotypes reflected different amounts of Sar1[T39N] expressed in a particular cell. When pRT-GFP-8K and pRT-12K/8K were co-expressed with Sar1[T39N] and ST-YFP, GFP fluorescence was associated with typical peripheral bodies, whereas the blockage of COPII-dependent transport in the same cell was easily detected by changes in ST-YFP localization (Fig. 4d
). These data show that the dominant negative mutant of Sar1 had no effect on TGBp3 intracellular transport, which could occur, therefore, in a COPII-independent manner. Importantly, co-bombardment of pRT-GFP-8K and full-length infectious cDNA clone pPVX201 with Sar1[T39N] also resulted in Sar1-independent intracellular GFP localization to peripheral bodies (Fig. 4f
).
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DISCUSSION |
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In the presence of native TGBp3, GFP-fused TGBp3 was found in peripheral bodies that represented membrane structures containing an ER marker (Fig. 3jl). Subcellular localization of PVX TGBp3 is similar to that of hordeiviral TGBp3, which has been previously shown to accumulate in ER-derived peripheral membrane bodies associated with plasmodesmata (Solovyev et al., 2000
; Zamyatnin et al., 2002
; Gorshkova et al., 2003
). Similar peripheral bodies were also induced by PMTV TGBp3 (Zamyatnin et al., 2004
; Haupt et al., 2005
). These observations strongly suggest that PVX TGBp3-containing peripheral bodies represent enlarged forms of structures functionally linked to the virus translocation pathway through plasmodesmata rather than simple inclusion bodies where superexpressed protein is deposited. Therefore, in spite of possible artefacts in the experimental system, which should always be kept in mind when working with GFP-fused protein expressed at high levels, such as mistargeting and misfolding of fusions or their detrimental effect on the functioning of the whole cell due to overexpression (Brandizzi et al., 2002
), the presented data on co-localization with the ER marker protein and observations of peripheral bodies in PVX-infected cells (Fig. 3b and c
) strongly support the functional relevance of these structures.
The potexviral TGBp3 consists of two structural elements, the N-terminal hydrophobic sequence, which potentially forms a transmembrane domain, and the C-terminal hydrophilic region containing a sequence motif conserved in the TGBp3 of representatives of the Flexiviridae (Fig. 1) (Morozov et al., 1991
; Adams et al., 2004
; Morozov & Solovyev, 2003
). The hydrophobic sequence is necessary for interaction with cell membranes and protein activity in cell-to-cell movement (Morozov et al., 1991
; Krishnamurthy et al., 2003
). Moreover, fusion of GFP to this N-terminal hydrophobic segment inhibits functional activity of PVX TGBp3 (see above). In this paper, the role of the protein hydrophilic C-terminal region in the functional competence of the protein was also analysed in complementation tests and targeting to cell peripheral compartments. For these experiments, three PVX TGBp3 mutants with C-terminal truncations of 24, 28 or 39 aa residues (8K
24, 8K
28 and 8K
39, respectively) were constructed (Fig. 1
). Note that the N-terminal transmembrane segment was intact in these mutants. In co-expression with PVX.GFP.
AUG8K, the TGBp3 mutants were functionally competent. They demonstrated a similar level of cell-to-cell movement complementation, although this was, however, lower than that of the wild-type TGBp3 (Fig. 2
).
Upon co-expression in epidermal N. benthamiana cells, both of the two C-terminally truncated TGBp3 mutants tested directed GFP-fused TGBp3 to the peripheral bodies, similar to the situation found with wild-type TGBp3 (Fig. 3h and i). We believe that at least part of the peripheral structures observed with the C-terminally truncated mutants are identical to the bodies directed by the wild-type protein because of the ability of mutant proteins to complement PVX.GFP.
AUG8K (see above). Therefore, one can conclude that these mutants have retained the ability of TGBp3 to complement cell-to-cell trafficking of movement-deficient PVX and to move to the peripheral bodies. Moreover, even complete deletion of the hydrophilic region of PVX TGBp3 (mutant 8K
39) did not block the ability to direct GFP-fused TGBp3 to the peripheral sites. However, some peculiarities of intracellular sorting are likely to be perturbed by deletions in the PVX TGBp3 hydrophilic region. In co-bombardment experiments, such deletion variants induced formation of large amorphous TGBp2-containing inclusions in the cell interior in addition to typical peripheral bodies (our unpublished data).
Our data suggest that indispensable signals for intracellular protein trafficking to the peripheral compartments are located in the N-terminal transmembrane hydrophobic sequence of PVX TGBp3. Similarly, translocation of hordeiviral TGBp3 to the cell periphery requires a signal in its C-terminal hydrophobic sequence (Solovyev et al., 2000; our unpublished data). Also, trafficking and localization of some cell membrane proteins were found to be determined by the composition and the length of their hydrophobic sequences (particularly the N-terminal hydrophobic segment) (Rayner & Pelham, 1997
; Letourneur & Cosson, 1998
; Reggiori et al., 2000
; Szczesna-Skorupa & Kemper, 2001
; Watson & Pessin, 2001
; Dirnberger et al., 2002
; Goder & Spiess, 2003
).
Further studies are required to resolve the molecular details of the TGBp3 N-terminal transmembrane region-specified translocation pathway to peripheral bodies. As a rule, transport of membrane-anchored proteins from the sites of their synthesis in the ER involves COPII-coated transport vesicles, which bud from ER membranes in ER exit sites (ERES) distributed throughout the cell and later fuse to the Golgi (Nebenführ, 2002; Barlowe, 2003
; van Vliet et al., 2003
; daSilva et al., 2004
; Hawes, 2005
). Budding of the COPII vesicles requires a small GTPase, Sar1, which initiates the formation of budding membrane complexes containing cargo proteins (van Vliet et al., 2003
). To get an insight into the TGBp3 trafficking pathway, a known method was used to disrupt the ER exit of proteins by co-expression with a dominant-negative GDP-restricted mutant of GTPase Sar1, Sar1[T39N], which prevents COPII prebudding complex formation (Andreeva et al., 2000
; Brandizzi et al., 2004
). Sar1[T39N] efficiently blocked the ER exit of ST-YFP, a control Golgi marker (Fig. 4b and c
) (Neumann et al., 2003
; Brandizzi et al., 2004
). However, GFP-tagged PVX TGBp3 reached peripheral bodies in the presence of wild-type TGBp3 in spite of blockage of the COPII-dependent transport by Sar1[T39N] (Fig. 4d
). Thus, the intracellular transport of TGBp3 to peripheral bodies involves a COPII-independent pathway. Indeed, evidence has been provided for the existence of such non-classical trafficking systems for protein transport to the plasma membrane and vacuoles (Mitsuhashi et al., 2001
; Törmäkangas et al., 2001
; Neumann et al., 2003
; Siddiqi et al., 2003
; Delmas et al., 2004
; Tamura et al., 2004
). Unfortunately, only very limited information is available on the molecular mechanisms that target cargo proteins to non-conventional ER export routes (Nebenführ, 2002
; Barlowe, 2003
; Bonifacino & Lippincott-Schwartz, 2003
). Interestingly, the ability of TGBp3 to enter the COPII-independent trafficking pathway is blocked by the short C-terminal truncation in 8K
24 (Fig. 4e
). So, this mutant may only enter the conventional COPII-dependent trafficking pathway from the ER. It is conceivable that the wild-type TGBp3 may use both COPII-independent and common COPII-dependent pathways.
The data presented here allow us to propose a model for the TGBp3 trafficking mechanism and TGBp3-directed movement of viral RNA and proteins. According to this model (Fig. 5), TGBp3 can accumulate in small membrane vesicles formed on the ER membrane in a Sar1-independent manner (Fig. 5a
, step 1a) and then fuse to Golgi stacks (Fig. 5a
, step 1b), as documented for the general pathway of membrane protein trafficking (van Vliet et al., 2003
). Also, TGBp3 can reach the Golgi without formation of discrete transport vesicles via a newly discovered mechanism of Sar1-dependent sliding of ERES together with individual Golgi stacks along the ER membrane (daSilva et al., 2004
; Hawes, 2005
) (Fig. 5a
, step 1b'). In both cases, further TGBp3 transport to plasmodesmata occurs via Golgi movement along actin filaments (Fig. 5a
, step 1c) and further formation of Golgi-derived vesicles (Fig. 5a
, step 1d) (Hawes, 2005
). Alternatively, the TGBp3 can be delivered to plasmodesmata via a Golgi-independent pathway in ER-derived transport vesicles that are probably transported to plasmodesmal orifices along actin filaments (Fig. 5a
, steps 2a and 2b) (Roberts & Oparka, 2003
; Oparka, 2004
; Haupt et al., 2005
). Whatever the origin of the TGBp3-specific transport vesicles, these carriers serve to deliver TGBp2, TGBp1 and viral RNA to plasmodesmata-associated sites using targeting signal(s) in TGBp3 (Solovyev et al., 2000
; Zamyatnin et al., 2002
; 2004
; Gorshkova et al., 2003
; Morozov & Solovyev, 2003
; Oparka, 2004
; Haupt et al., 2005
). TGBp2 and TGBp3 enter the ER co-translationally due to integration of their hydrophobic regions into the ER membrane. Importantly, translation of TGBp2 and TGBp3 is co-ordinated in the process of reading a single subgenomic RNA (Morozov & Solovyev, 2003
), which might provide a mechanism for TGBp2 and TGBp3 co-segregation for ER exit in the same transport vesicles. TGBp2/TGBp3-containing vesicles (or Golgi stacks) bind to movement-competent virions (or RNPs) through proteinprotein interactions and/or by direct association with RNA (Morozov & Solovyev, 2003
).
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ACKNOWLEDGEMENTS |
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Received 28 December 2004;
accepted 24 March 2005.