Cell‐to‐cell transport of proteins: requirement for unfolding and characterization of binding to a putative plasmodesmal receptor

Abstract

SummaryPlasmodesmata and the nuclear pore complex (NPC) mediate the selective trafficking of proteins and protein-nucleic acid complexes. The events underlying the translocation of endogenous and viral proteins through plasmodesmata were investigated to further explore the parallels between these cell-to-cell and intracellular communication systems. Studies performed with crosslinked KNOTTED1 (KN1) revealed that a conformational change is required for the cell-to-cell movement of this protein. Microinjection of gold-conjugated KN1 established that, as with the NPC, a combination of protein unfolding and microchannel dilation appears to be involved in protein translocation. However, during this process the extent of microchannel dilation is much less than observed for the NPC, which may reflect a physical limitation imposed by the cell wall. Co-injection of KN1-gold with unbound KN1 or cucumber mosaic virus movement protein (CMV-MP) established that the KN1-gold probe is highly effective at blocking plasmodesmal transport of KN1 and CMV-MP. This result provided the foundation for competition experiments which demonstrated that KN1 and the viral movement proteins of CMV and tobacco mosaic virus likely utilize a common receptor in the pathway for cell-to-cell transport of proteins. A combination of biochemical fractionation methods, an in vitro binding assay founded on the high affinity between KN1-gold and the putative common plasmodesmal receptor, and microinjection techniques were used to isolate plasmodesmal constituents involved in cell-to-cell transport. A model describing the steps involved in protein transport through plasmodesmata is presented.

Developmental regulation and significance of KNOX protein trafficking inArabidopsis

“…So far, studies using viral MPs and rice phloem proteins suggest that recognition by the PD trafficking machinery involves structural motifs (Haywood et al, 2002;Ishiwatari et al, 1998), though a short sequence motif appears to control the trafficking of the heat shock cognate 70 chaperone (Aoki et al, 2002). In the case of KN1, a short peptide, homologous to a region near the N terminus of the protein, can interfere with its trafficking, though it is not known whether this sequence motif is sufficient for trafficking (Kragler et al, 1998).…”

“…One possible mechanism would be if KN1 was modified and gained a novel function during intercellular movement. Partial unfolding of KN1 is required for passage through PDs (Kragler et al, 1998), and could expose KN1 to post-translational modification(s). Alternatively, the lack of a KNOX overexpression phenotype in the 35S-GUS~KN1 plants could be because the fusion of GUS inhibits the ability KN1 to interact with partner proteins, such as Arabidopsis homologs of KN1 interacting protein (KIP), a BEL1-like TALE homeodomain protein (Smith et al, 2002).…”

MPB2C, a Microtubule-Associated Plant Protein Binds to and Interferes with Cell-to-Cell Transport of Tobacco Mosaic Virus Movement Protein

“…Tag-MPB2C lib was overexpressed from plasmid pRSETA-⌬1-197MPB2C, and western-blot analysis of tag-MPB2C lib was done according to the manufacturer's protocol (Invitrogen, Carlsbad, CA). Conditions for renatured blot overlay assay are based on previously described dot-blot assays (Kragler et al, 1998). In brief, MP30 and CMV3a were blotted onto nitrocellulose membranes, treated twice for 15 min at room temperature with incubation buffer (40 mm HEPES, pH 6.8, and 10% [v/v] glycerol) containing 0.2% (v/v) Triton X-100 and Complete Protease Inhibitor Cocktail Tablets (Roche Diagnostics).…”

“…So far, a cell wall-associated pectin methyl esterase (PME; Dorokhov et al, 1999;Tzfira et al, 2000) was shown to specifically interact with TMV-MP30. Also, binding studies of MP30 with cell fractions enriched in plasmodesmal components indicate the presence of potential receptor molecules mediating protein movement via plasmodesmata (Kragler et al, 1998). Recently, Nt-NCAPP1, an ER-localized protein, was found to be involved in the plasmodesmal transport pathway of MP30 (Lee et al, 2003).…”

Different subcellular localization and trafficking properties of KNOX class 1 homeodomain proteins from rice

“…Specific proteins have the capacity to dilate plasmodesmata, enabling large proteins, ribonucleoprotein complexes and viral particles to pass through (Lucas, 1999). Dilation of plasmodesmata is an active process triggered by the protein that is to be transported, and is reversed after its passage (Kragler et al, 1998). The plasmodesmal transport pathway appears to involve specific interactions between the non-cell-autonomous proteins and the plasmodesma, but the complexity of plasmodesmal regulation is still far from understood.…”