Previous work on TGB-mediated viral cell-to-cell movement suggested that the endomembrane-anchored viral movement protein TGBp3 is the key determinant of the intracellular transport of a viral genome to plasmodesmata (41
). Here we confirm that PSLV TGBp3 colocalizes with TMV p30 and BYV Hsp70h, two unrelated viral proteins that are autonomously targeted to plasmodesmata (Fig. ) (39
). Recent work demonstrated that the proper targeting of Hsp70h requires the actin cytoskeleton (51
). In addition, actin microfilaments or microtubules were implicated in the intracellular transport of several other viral MPs (2
). Unexpectedly, our analysis demonstrated that the drugs that specifically disassemble or stabilize either microfilaments or microtubules had no observable effect on the targeting of either PSLV or PVX TGBp3 to plasmodesmata (Fig. and ). These results indicated that the localization of diverse TGBp3 variants is independent of cytoskeletal motility systems and is therefore mechanistically distinct from that of BYV Hsp70h.
Interestingly, the role of the cytoskeleton in the plasmodesmatal targeting of TMV p30 remains a matter of debate. One line of evidence based primarily on a genetic approach supports microtubule-based motility as the primary means of p30 targeting (9
). In contrast, the combination of the pharmaceutical approach and fluorescence recovery after photobleaching suggested a role for the actin microfilaments but not the microtubules in p30 localization (66
). In our hands, neither actin- nor tubulin-depolymerizing drugs affected p30 accumulation in plasmodesmata (51
). It seems likely that the apparent discrepancy between the results of these studies is due to different experimental designs. Indeed, the first study examined a correlation between p30 association with microtubules and TMV movement (9
), the second investigated the dynamics of p30 accumulation in the plasmodesmata of TMV-infected cells (66
), and our study addressed the autonomous targeting of p30 in noninfected cells (51
Because PSLV TGBp3 is tightly associated with the ER-derived membranes, we were also interested in exploring the possible involvement of vesicular trafficking in TGBp3 delivery to plasmodesmata. As a rule, the intracellular transport of membrane-anchored and secreted proteins involves ER exit in COPII-coated vesicles, which bud from the ER and fuse to the Golgi apparatus (4
). We showed that GFP-tagged TGBp3 was able to reach the peripheral bodies in the plasmodesmatal vicinity when the exit of COPII-dependent protein from the ER was suppressed by Sar1(T34N) (Fig. ), the dominant negative Sar1 mutant which prevents COPII budding complex formation (1
). In addition, TGBp3 targeting was insensitive to BFA, which blocks the Golgi body-dependent secretory pathway and induces Golgi body fusion to the ER (Fig. ). Because the movement of Golgi stacks and transport vesicles in plant cells depends on actin microfilaments (7
), the results of our experiments targeting the cytoskeleton and vesicular trafficking are in agreement with each other. Taken together, these data strongly argue against the vesicular model of TGBp3 intracellular transport. We therefore propose that TGBp3 transport occurs via an unconventional pathway that is independent of transport vesicles and the cytoskeleton. In accord with a recent study (66
), we found that the vesicular secretion pathway is not required for the plasmodesmatal targeting of TMV p30 (Fig. ), suggesting that this ER-associated MP may also use an unconventional transport pathway.
We further used mutational analysis to identify the determinants that specify an unusual transport pathway for PSLV TGBp3 to the plasmodesmata. Two sequence elements, the central hydrophilic region containing the conserved pentapeptide YQDLN and the putative C-terminal transmembrane domain, were found to form a composite signal that was both essential and sufficient for targeting PSLV TGBp3 to plasmodesmata. In accord with previous work (10
), we also found that the length of the membrane-spanning domain is critical for the proper localization of TGBp3 (Fig. ). Finally, we showed that this domain should be at the very C terminus of the protein in order to target TGBp3 to the plasmodesmata. It should be emphasized that the mapping of localization determinants to specific regions of TGBp3 supports the functional role of these determinants in their interaction with an endogenous trafficking pathway.
What is the possible mechanism of TGBp3 targeting to plasmodesmata? Since TGBp3 lacks the N-terminal signal peptide, its targeting to membranes is most likely posttranslational, which is rather typical of the tail-anchored proteins (5
). One example of such a protein is provided by the yeast integral membrane protein Ist2p, which is sorted to the plasma membrane by translocon-, Golgi body-, and actin microfilament-independent mechanisms (27
). It was proposed that Ist2p moves intracellularly by the diffusion or local fusion of the ER and plasma membrane. Another parallel can be drawn from the plant Rop7 protein, a small, membrane-anchored, Ras-related GTPase that requires neither the cytoskeleton nor Golgi stacks for its subcellular targeting (3
). By analogy with these plasma membrane-targeted proteins, we hypothesize that TGBp3 traffics to its distinct cellular destination, the plasmodesmatal vicinity, via diffusion in the cytoplasm, possibly by forming a complex with the cell chaperones. The directionality of the TGBp3 transport may be explained by a diffusion-and-retention mechanism, whereby TGBp3 is anchored upon reaching the plasmodesmata. Such anchoring may involve protein-protein interactions mediated by the hydrophilic region adjacent to the C-terminal membrane-spanning domain of TGBp3. The experimental confirmation of this hypothetical model can be obtained by tracing the TGBp3 trafficking pathway using single-molecule imaging techniques. In general, our work highlights the diversity of the cellular trafficking pathways that are recruited by viral proteins to aid their delivery to target cell compartments such as plasmodesmata.