Patellin1, a novel Sec14-like protein, localizes to the cell plate and binds phosphoinositides

Abstract

Membrane trafficking is central to construction of the cell plate during plant cytokinesis. Consequently, a detailed understanding of the process depends on the characterization of molecules that function in the formation, transport, targeting, and fusion of membrane vesicles to the developing plate, as well as those that participate in its consolidation and maturation into a fully functional partition. Here we report the initial biochemical and functional characterization of patellin1 (PATL1), a novel cell-plate-associated protein that is related in sequence to proteins involved in membrane trafficking in other eukaryotes. Analysis of the Arabidopsis genome indicated that PATL1 is one of a small family of Arabidopsis proteins, characterized by a variable N-terminal domain followed by two domains found in other membrane-trafficking proteins (Sec14 and Golgi dynamics domains). Results from immunolocalization and biochemical fractionation studies suggested that PATL1 is recruited from the cytoplasm to the expanding and maturing cell plate. In vesicle-binding assays, PATL1 bound to specific phosphoinositides, important regulators of membrane trafficking, with a preference for phosphatidylinositol(5)P, phosphatidylinositol(4,5)P(2), and phosphatidylinositol(3)P. Taken together, these findings suggest a role for PATL1 in membrane-trafficking events associated with cell-plate expansion or maturation and point to the involvement of phosphoinositides in cell-plate biogenesis.

Figure 1.

Arabidopsis PATL protein structures. A, Diagram of the domain structure of Arabidopsis PATLs. The percentage amino acid identity/similarity between the Sec14 and GOLD domains of PATL1 and the other patellin family members is shown in parentheses below the domains. Pairwise analysis was performed using ClustalW with a gap opening penalty of 10 and a gap extension penalty of 0.2. The molecular masses predicted from the open reading frame sequences are shown. B, Amino acid sequence alignments of the Sec14 domain of the Arabidopsis PATLs and yeast Sec14p. Sec14p residues involved in PtdIns transfer activity are marked with a star, while hydrophobic residues that line the lipid-binding pocket are indicated by arrows. C, Amino acid sequence alignments of the C-terminal Lys-rich motif of the PATLs.

Figure 2.

PATL1 antibody production and characterization. The Arabidopsis PATL1 coding region was expressed as a GST-fusion in E. coli and purified for biochemical analysis and antibody production. A, SDS-PAGE of uninduced (lane 1) and induced (lane 2) E. coli extracts, purified GST-patellin (lane 3), and cleaved fusion protein (lane 4). Molecular mass markers are shown at left. B, Immunoblots of Arabidopsis root total protein extracts (10 μg/lane) probed with PATL1 antisera raised against full-length protein (lane 1) or the GOLD domain (lane 2). C, Immunoblots of PATL1 and PATL2-GST fusion proteins probed with PATL1 antibody. D, Immunoblots of Arabidopsis organ extracts (10 μg/lane) probed with PATL1 or actin antibodies.

Figure 3.

PATL1 is a peripheral membrane protein. A, Immunoblots of Arabidopsis root subcellular fractions. Arabidopsis root extracts were centrifuged at 1,000g to yield a total cell extract (S1). The S1 fraction was centrifuged at 100,000g to yield soluble (S100) and membrane (P100) fractions. The S1, S100, and P100 fractions were analyzed by immunoblotting with antibodies to PATL1 and the integral membrane marker SEC12. Similar results were obtained in three independent experiments. B, Samples of the P100 fraction were washed in the indicated solutions. A total sample (T) was collected prior to centrifugation at 100,000g to yield the solubilized protein (S). Membrane equivalents of the T and S samples were analyzed by immunoblotting with antibodies to PATL1 or SEC12. Similar results were obtained in two independent experiments.

Figure 4.

Immunolocalization of PATL1 in Arabidopsis roots and tobacco BY2 cells. Arabidopsis roots were immunolabeled and imaged by confocal microscopy. A, Confocal projection compiled from 1 μm sections through an Arabidopsis root tip shows cell-plate labeling for PATL1 (green) in the zone of cell division. B, Single optical section from the projection shown in A shows PATL1 staining of cell plates and punctate cytoplasmic structures. Bars in A and B = 20 μm. C, Colocalization of PATL1 (green) with tubulin (red) and DNA (blue) in a file of cortical cells. Arrowheads mark the sites of PATL1 staining of expanding cell plates. The asterisk marks a cell with a preprophase band. D, Interphase cells stained for PATL1 (green). E, Interphase cells expressing Golgi-associated GmMan1::GFP (green) and immunostained for PATL1 (red). F to L, Colocalization of PATL1 (green) with tubulin (red) and DNA (blue). Single optical sections midway through the division plane are shown in F to J. Interphase (D) and metaphase (F) cells show PATL1 labeling of 1 μm cytoplasmic structures (marked with arrowheads). Early- (G), late- (H), and post- (I) telophase cells show PATL1 staining of the developing cell plate. Three-dimensional computer reconstructions built from 0.2-μm sections through the top half of the phragmoplast shown in J are projected at 10° and 20° from the optical axis in K and L, respectively. These images show that PATL1 at the plate is fibrillar in structure. Bars in D to L = 10 μm.

Figure 5.

PATL1 binds phosphoinositides. Vesicle cosedimentation assays were used to test PATL1 protein for phospholipid-binding activity. A, PATL1 (2.5 μg) was mixed with unilamellar vesicles composed of 25 mm PtdEth, PtdCh, or PtdIns. Vesicle bound proteins (P) were separated from unbound proteins (S) by centrifugation at 164,000g and analyzed by fluorescence scanning of SYPRO red-stained SDS gels. PATL1 protein alone was used as a blank. B, PATL1 binding to phosphoinositides was tested as described above except that unilamellar vesicles were made from a mixture of 10:1 PtdEth:test phosphoinositide and the lipid concentration was 2.2 mm. The percentage of patellin bound was quantified using ImageQuant software. Values are the mean ± se from at least three different experiments.