A subclass of plant heat shock cognate 70 chaperones carries a motif that facilitates trafficking through plasmodesmata

Abstract

Plasmodesmata establish a pathway for the trafficking of non-cell-autonomously acting proteins and ribonucleoprotein complexes. Plasmodesmal enriched cell fractions and the contents of enucleate sieve elements, in the form of phloem sap, were used to isolate and characterize heat shock cognate 70 (Hsc70) chaperones associated with this cell-to-cell transport pathway. Three Cucurbita maxima Hsc70 chaperones were cloned and functional and sequence analysis led to the identification of a previously uncharacterized subclass of non-cell-autonomous chaperones. The highly conserved nature of the heat shock protein 70 (Hsp70) family, in conjunction with mutant analysis, permitted the characterization of a motif that allows these Hsc70 chaperones to engage the plasmodesmal non-cell-autonomous translocation machinery. Proof of concept that this motif is necessary for Hsp70 gain-of-movement function was obtained through the engineering of a human Hsp70 that acquired the capacity to traffic through plasmodesmata. These results are discussed in terms of the roles likely played by this subclass of Hsc70 chaperones in the trafficking of non-cell-autonomous proteins.

Fig 1.

Immunodetection of heat shock protein homologs in plasmodesmal enriched protein fractions and vascular tissues. (A) Plasmodesmal enriched protein fractions were prepared from tobacco tissue (W2) and BY-2 tobacco suspension cultured cells (PECP), and aliquots (10 μg) were separated by SDS/PAGE and subjected to Western analysis to test for the presence of Hsp70 homologs. (B–D) Immunodetection of Hsp70 and Hsp40 homologs in proteins prepared from C. maxima (pumpkin) stem tissues, vascular bundles, and phloem sap. (B) Protein samples (30 μg) were separated by SDS/PAGE and stained with GelCode Blue Reagent. (C and D) Detection of Hsp70 and Hsp40 homologs in protein fractions shown in B. Western analysis was performed with anti-wheat Hsp70 (A and C) and anti-cucumber CsDnaJ-1 polycolonal antibodies (D); control blots were performed in the absence of secondary antibodies.

Fig 2.

Authentication of CmHsc70-1 and CmHsc70-2 as phloem sap proteins by using peptide mass fingerprint analysis. (A) Phloem sap Hsp70 isoforms were purified to near homogeneity by chromatographic separation of phloem sap using a combination of HiTrap Q-Sepharose (Q-Sepharose) and C8-linked ATP-agarose (ATP-agarose) columns. Trypsin-digestion of the ATP-agarose purified 70-kDa protein band generated peptides that could be assigned to either CmHsc70-1 or CmHsc70-2, but not to CmHsc70-3. (B) Chromatographic separation of the ATP-agarose fractionated phloem proteins on a Mono Q-Sepharose 5/5R column. Fractions were quantified by densitometry and individual bands were excised for mass fingerprint analysis.

Fig 3.

Recombinant Hsc70 proteins exhibit ATPase activity. (A) His-6-tagged CmHsc70-1, CmHsc70-2, CmHsc70-3, and DnaK purified by using Ni-based chromatography. The CmHsc40-1 cochaperone was purified as a GST fusion protein. (B) Steady-state ATP-hydrolytic activity of recombinant His-6-tagged Hsc70 isoforms (7 μg) measured alone or in the presence of GST (2.6 μg) or GST-CmHsc40-1 (7 μg). Bars represent the mean (±SD) for three independent measurements and are based on six sample points collected during the linear phase of the reaction.

Fig 4.

Phloem CmHsc70 isoforms contain a motif necessary and sufficient to mediate their cell-to-cell movement. (A Upper) The conserved structural organization of the Hsp70 family. The N-terminal 392 amino acid residues correspond to the ATPase domain; residues 393–604 represent the region of the protein involved in substrate/peptide binding, which is comprised of β-sandwich and α-helical subdomains. The C terminus is divided into a SVR and the TPR–PB. Movement capacity of wild-type and mutant isoforms of CmHsc70-1, CmHsc70-2, CmHsc70-3, and human Hsp70 was determined by standard microinjection methods (21, 27). The capacity of each probe to move cell to cell was evaluated against CmPP16 (27) that was injected into the same leaf tissues. Probes that moved into >20 cells were characterized as highly competent (+++); those that moved into 15–20 cells were characterized as very competent (++), those that moved into >7 cells were characterized as competent (+), and no movement was characterized as incompetent (−). (B) Comparative analysis of the C-terminal region of C. maxima, Arabidopsis thaliana, and Homo sapiens cytosolic Hsp70 chaperone families present in genomic databases (www.ncbi.nlm.nih.gov). An endoplasmic reticulum Hsp70 isoform, At5g28540, was included for comparison. Colors are as follows: green, polar; yellow, nonpolar; blue, basic; red, acidic residues.