Rice SCAMP1 Defines Clathrin-Coated, trans- Golgi–Located Tubular- Vesicular Structures as an Early Endosome in Tobacco BY-2 Cells

Abstract We recently identified multivesicular bodies (MVBs) as prevacuolar compartments (PVCs) in the secretory 分泌 and endocytic 内吞 pathways to the lytic 细胞溶解酶 vacuole in tobacco (Nicotiana tabacum) BY-2 cells. We recently identified multivesicular bodies (MVBs) as prevacuolar compartments (PVCs) in the secretory 分泌 and endocytic 内吞 pathways to the lytic 细胞溶解酶 vacuole in tobacco (Nicotiana tabacum) BY-2 cells. Secretory carrier membrane proteins (SCAMPs) 分泌载体膜蛋白 are post-Golgi, integral membrane proteins 整合蛋白 mediating endocytosis in animal cells. Secretory carrier membrane proteins (SCAMPs) 分泌载体膜蛋白 are post-Golgi, integral membrane proteins 整合蛋白 mediating endocytosis in animal cells.

To define the endocytic pathway in plants, we cloned the rice (Oryza sativa) homolog of animal SCAMP1 and generated transgenic tobacco BY-2 cells expressing yellow fluorescent protein (YFP)–SCAMP1 or SCAMP1-YFP fusions. To define the endocytic pathway in plants, we cloned the rice (Oryza sativa) homolog of animal SCAMP1 and generated transgenic tobacco BY-2 cells expressing yellow fluorescent protein (YFP)–SCAMP1 or SCAMP1-YFP fusions. Confocal 共焦的 immunofluorescence 免疫荧 光 and immunogold electron microscopy studies demonstrated that YFP-SCAMP1 fusions and native SCAMP1 localize to the plasma membrane and mobile structures in the cytoplasm of transgenic BY-2 cells. Confocal 共焦的 immunofluorescence 免疫荧 光 and immunogold electron microscopy studies demonstrated that YFP-SCAMP1 fusions and native SCAMP1 localize to the plasma membrane and mobile structures in the cytoplasm of transgenic BY-2 cells.

RESULTS

1.Highly Conserved Plant SCAMPs (A) Alignment of amino acid sequences of SCAMPs from the rice (Oryza sativa) cDNA clone used in this study (SCAMP cDNA); a pea (Pisum sativum) cDNA (Ps ), an Arabidopsis thaliana cDNA (At ), and an animal (Rattus norvegicus) SCAMP1 cDNA (Rn ). (A) Alignment of amino acid sequences of SCAMPs from the rice (Oryza sativa) cDNA clone used in this study (SCAMP cDNA); a pea (Pisum sativum) cDNA (Ps ), an Arabidopsis thaliana cDNA (At ), and an animal (Rattus norvegicus) SCAMP1 cDNA (Rn ).Oryza sativaPisum sativum Arabidopsis thalianaRattus norvegicusOryza sativaPisum sativum Arabidopsis thalianaRattus norvegicus

Gray areas indicate conserved amino acids among the four SCAMPs. Two synthetic peptides, corresponding to the N- terminal NPF motifs and the middle cytosolic loop, used in this study to generate antibodies are highlighted in boxes. The four transmembrane domain regions are underlined. The overall similarity among all known plant SCAMPs is >80% at the amino acid level. Gray areas indicate conserved amino acids among the four SCAMPs. Two synthetic peptides, corresponding to the N- terminal NPF motifs and the middle cytosolic loop, used in this study to generate antibodies are highlighted in boxes. The four transmembrane domain regions are underlined. The overall similarity among all known plant SCAMPs is >80% at the amino acid level.

(B) Predicted structure of the rice SCAMP1 used in this study. The predicted topology of this plant SCAMP1 consists of two NPF (Asn-Pro-Phe) repeats at its N-terminal region in the cytosol, four transmembrane domains (TMD), and a short cytosolic C- terminal region. (B) Predicted structure of the rice SCAMP1 used in this study. The predicted topology of this plant SCAMP1 consists of two NPF (Asn-Pro-Phe) repeats at its N-terminal region in the cytosol, four transmembrane domains (TMD), and a short cytosolic C- terminal region.

2. Generation of Transgenic SCAMP1-YFP BY-2 Cells and Characterization of SCAMP1 Antibodies (A) Top panel, chimeric DNA constructs. YFP was fused at either the N terminus of SCAMP1 (YFP-SCAMP) or the C terminus of SCAMP1 (SCAMP- YFP). The fusions were expressed under the control of the cauliflower mosaic virus 35S promoter and the 3′ NOS terminator. (A) Top panel, chimeric DNA constructs. YFP was fused at either the N terminus of SCAMP1 (YFP-SCAMP) or the C terminus of SCAMP1 (SCAMP- YFP). The fusions were expressed under the control of the cauliflower mosaic virus 35S promoter and the 3′ NOS terminator.

Bottom panel, protein gel blot analysis of transgenic tobacco BY-2 cell lines. Soluble protein (CS) and membrane protein (CM) were isolated from both wild-type and transgenic BY-2 cells, followed by protein separation via SDS- PAGE and protein detection using GFP antibodies. The asterisk indicates the position of the expressed full-length YFP fusion protein. Bottom panel, protein gel blot analysis of transgenic tobacco BY-2 cell lines. Soluble protein (CS) and membrane protein (CM) were isolated from both wild-type and transgenic BY-2 cells, followed by protein separation via SDS- PAGE and protein detection using GFP antibodies. The asterisk indicates the position of the expressed full-length YFP fusion protein.

(B) Characterization of SCAMP1a antibodies. Soluble (CS) and membrane (CM) proteins were isolated from wild- type BY-2 cells, followed by SDS-PAGE and protein gel blot detection with anti-SCAMP1a. The asterisk indicates the position of SCAMP1. M, molecular mass in kilodaltons. (B) Characterization of SCAMP1a antibodies. Soluble (CS) and membrane (CM) proteins were isolated from wild- type BY-2 cells, followed by SDS-PAGE and protein gel blot detection with anti-SCAMP1a. The asterisk indicates the position of SCAMP1. M, molecular mass in kilodaltons.

(C) Protein gel blot analysis of transgenic tobacco BY-2 cell lines overexpressing SCAMP1. Proteins were extracted from the wild type (lane 1) and several transgenic BY-2 cell lines overexpressing SCAMP1 (lanes 2 to 7), followed by SDS-PAGE and protein gel blot detection with anti- SCAMP1b. The asterisk indicates the position of SCAMP1. M, molecular mass in kilodaltons. (C) Protein gel blot analysis of transgenic tobacco BY-2 cell lines overexpressing SCAMP1. Proteins were extracted from the wild type (lane 1) and several transgenic BY-2 cell lines overexpressing SCAMP1 (lanes 2 to 7), followed by SDS-PAGE and protein gel blot detection with anti- SCAMP1b. The asterisk indicates the position of SCAMP1. M, molecular mass in kilodaltons.

(D) Subcellular localization of SCAMP1 in wild-type cells (panels 1 and 3) and transgenic BY-2 cells overexpressing SCAMP1 (panels 2 and 4) via confocal immunofluorescence with SCAMP1 antibodies as indicated. Arrowheads indicate examples of PM localization of punctate signals. (D) Subcellular localization of SCAMP1 in wild-type cells (panels 1 and 3) and transgenic BY-2 cells overexpressing SCAMP1 (panels 2 and 4) via confocal immunofluorescence with SCAMP1 antibodies as indicated. Arrowheads indicate examples of PM localization of punctate signals.

3.Subcellular Localization of the SCAMP1-YFP Fusion Construct and SCAMP1 in BY-2 Cells Subcellular Localization of YFP-SCAMP1 Fusion Constructs in Transgenic Tobacco BY-2 Cells. Subcellular Localization of YFP-SCAMP1 Fusion Constructs in Transgenic Tobacco BY-2 Cells. Confocal images of YFP signals in cells expressing either YFP-SCAMP1 fusion construct were collected from untreated cells (panel 1), protoplasts (panel 2), and cells treated with 1.5 M NaCl 2 (panel 3). Confocal images of YFP signals in cells expressing either YFP-SCAMP1 fusion construct were collected from untreated cells (panel 1), protoplasts (panel 2), and cells treated with 1.5 M NaCl 2 (panel 3).

Subcellular Localization of SCAMP1 in Tobacco BY-2 Cells Panel 1, colocalization of SCAMP1a and SCAMP1b antibodies in wild-type BY-2 cells; Panel 1, colocalization of SCAMP1a and SCAMP1b antibodies in wild-type BY-2 cells; panel 2, colocalization of SCAMP1-YFP with anti-SCAMP1 in transgenic BY-2 cells; panel 2, colocalization of SCAMP1-YFP with anti-SCAMP1 in transgenic BY-2 cells; panel 3, colocalization of YFP-SCAMP with anti-SCAMP1 in transgenic BY-2 cells; panel 3, colocalization of YFP-SCAMP with anti-SCAMP1 in transgenic BY-2 cells;