The receptor kinases LePRK1 and LePRK2 associate in pollen and when expressed in yeast, but dissociate in the presence of style extract

Abstract

After pollen grains germinate on the stigma, pollen tubes traverse the extracellular matrix of the style on their way to the ovules. We previously characterized two pollen-specific, receptor-like kinases, LePRK1 and LePRK2, from tomato (Lycopersicon esculentum). Their structure and immunolocalization pattern and the specific dephosphorylation of LePRK2 suggested that these kinases might interact with signaling molecules in the style extracellular matrix. Here, we show that LePRK1 and LePRK2 can be coimmunoprecipitated from pollen or when expressed together in yeast. In yeast, their association requires LePRK2 kinase activity. In pollen, LePRK1 and LePRK2 are found in an approximately 400-kDa protein complex that persists on pollen germination, but this complex is disrupted when pollen is germinated in vitro in the presence of style extract. In yeast, the addition of style extract also disrupts the interaction between LePRK1 and LePRK2. Fractionation of the style extract reveals that the disruption activity is enriched in the 3- to 10-kDa fraction. A component(s) in this fraction also is responsible for the specific dephosphorylation of LePRK2. The style component(s) that dephosphorylates LePRK2 is likely to be a heat-stable peptide that is present in exudate from the style. The generally accepted model of receptor kinase signaling involves binding of a ligand to extracellular domains of receptor kinases and subsequent activation of the signaling pathway by receptor autophosphorylation. In contrast to this typical scenario, we propose that a putative style ligand transduces the signal in pollen tubes by triggering the specific dephosphorylation of LePRK2, followed by dissociation of the LePRK complex.

Fig. 1.

LePRK1 and LePRK2 associate with each other in pollen and in yeast membranes. (A) Membrane proteins from pollen (first and fourth lanes) were immunoprecipitated (second and third lanes) by using anti-ECD2 antibody. The proteins were separated by SDS/PAGE, and immunoblots were developed with anti-ECD1 antibody (first and second lanes) and anti-ECD2 antibody (third and fourth lanes). (B) Membrane proteins from yeast expressing LePRK1 and LePRK2 were immunoprecipitated by using anti-ECD2 antibody. The precipitated proteins were subjected to SDS/PAGE, and the immunoblot was developed with both anti-ECD1 and anti-ECD2 antibodies (fourth lane). First lane, yeast membrane preparations (P₁₀₀) expressing LePRK1; second lane, yeast membrane preparations expressing LePRK2; third lane, yeast membrane preparations expressing LePRK1 and LePRK2. (C) LePRK2 kinase activity is required for complex formation. Membrane proteins from yeast expressing LePRK1 and LePRK2 (first lane), LePRK1-(K396R) and LePRK2 (second lane), and LePRK1 and LePRK2-(K372R) (third lane) were immunoprecipitated by using anti-ECD2 antibody. The precipitated proteins were subjected to SDS/PAGE, transferred to membranes, and immunoblotted with both anti-ECD1 and anti-ECD2 antibodies.

Fig. 2.

LePRK1 and LePRK2 exist in pollen extract as oligomeric complexes that are dissociated by tomato style extract. Gel-filtration fractions of the Superdex 200 HR from mature pollen (A) and pollen germinated in the presence of style extract (B and C) were collected and separated by SDS/PAGE. The presence of LePRK1 and LePRK2 was determined by immunoblot analysis. The positions of the molecular mass standards are indicated. Numbers indicate the fraction number as eluted sequentially.

Fig. 3.

Tomato style extract disrupts the association of LePRK1 and LePRK2 in yeast. The first and second lanes correspond to membrane proteins (P₁₀₀) from yeast expressing LePRK1 and LePRK2, which had been incubated in the absence (first lane; -) or presence (second lane; SE) of tomato style extract. Duplicate samples (third and fourth lanes) were immunoprecipitated with anti-ECD2 antibody. The proteins were subjected to SDS/PAGE, transferred to membranes, and immunoblotted with both anti-ECD1 and anti-ECD2 antibodies.

Fig. 4.

The effective style extract component contains 3- to 10-kDa molecules. Style extract was loaded on YM10 filters (cutoff, 10 kDa), and the eluate (<10 kDa) was loaded on YM3 filters (cutoff, 3 kDa). After centrifugation, the retentate of YM10 (>10 kDa), the retentate of YM3 (3–10 kDa), and the eluate of YM3 (<3 kDa) were assayed for the ability to disrupt the association of LePRK1 and LePRK2 in yeast (A) and for the ability to dephosphorylate LePRK2 in pollen (B). The position of LePRK2 is indicated by arrows. In B, the LePRK2 on the IB precisely aligned with the upper band on the radiography film, and the lower bands did not align.

Fig. 5.

Style factor is likely to be a heat-stable peptide that is present in the style exudate. (A) Different amounts of total style extract were used to dephosphorylate LePRK2. (B) Pollen membranes (P₁₀₀) were incubated in phosphorylation buffer with [γ-³²]ATP in the absence (first lane; -) or presence (second lane; style exudate) of tobacco style exudate (20 μg of protein). Total proteins were separated by SDS/PAGE, blotted to nitrocellulose, and then subjected to autoradiography. The position of LePRK2 is indicated by an arrow. (C) Total tobacco style extract first was subjected to 95°C for 3 min (second lane) and 10 min (third lane) and then assayed for dephosphorylation of LePRK2. The position of LePRK2 is indicated by an arrow. (D) Total tobacco style extract (100 μg of protein) first was subjected to 95°C for 10 min (second lane) and then assayed for the ability to disrupt the association of LePRK1 and LePRK2 in yeast. (E) Total style extract (100 μg of protein) first was incubated or not with pronase (50 μg; 2.5 μg/μl)for 3h at 37°C, subjected to 95°C for 10 min, and then assayed for dephosphorylation of LePRK2. For A and E, the relative amounts of labeled LePRK2 in each treatment were estimated by scanning the gel with a Storm 820 PhosphorImager (Molecular Dynamics), and the values obtained were compared with signal of the treatment without style extract (-) as reference.

Fig. 6.

Model for LePRK1–LePRK2 signaling. LePRK1 and LePRK2 associate in mature pollen membranes (pregermination) as part of a multimeric protein complex (Left). In this complex, LAT52 interacts as an extracellular partner and hypothetical pollen proteins (X, Y, and Z) interact with the cytoplasmic domains of LePRK1 and of phosphorylated LePRK2. Upon germination on the stigma, the still unknown pistil ligand displaces LAT52 and, on binding, induces the dephosphorylation of LePRK2 (Center). The LePRK complex then dissociates, releasing the cytoplasmic partners (X, Y, and Z) and transducing the signal to the pollen tube (Right).