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

Abstract Abstract We recently identified multivesicular bodies We recently identified multivesicular bodies

(MVBs)(MVBs) as prevacuolar compartments as prevacuolar compartments (PVCs)(PVCs) in the secretoryin the secretory分泌分泌 and endocyticand endocytic内吞内吞 pathways to the lyticpathways to the lytic 细胞溶解酶细胞溶解酶vacuole in tobacco (vacuole in tobacco (Nicotiana tabacumNicotiana tabacum) BY-) BY-2 cells. 2 cells.

Secretory carrier membrane proteins Secretory carrier membrane proteins (SCAMPs)(SCAMPs)分泌载体膜蛋白分泌载体膜蛋白 are post-Golgi, are post-Golgi, integral membrane proteinsintegral membrane proteins 整合蛋白整合蛋白mediating endocytosis in animal cells. mediating endocytosis in animal cells.

To define the endocytic pathway in plants, we To define the endocytic pathway in plants, we cloned the rice (cloned the rice (Oryza sativaOryza sativa) homolog of animal ) homolog of animal SCAMP1 and generated transgenic tobacco BY-2 SCAMP1 and generated transgenic tobacco BY-2 cells expressing yellow fluorescent protein (YFP)–cells expressing yellow fluorescent protein (YFP)–SCAMP1 or SCAMP1-YFP fusions. SCAMP1 or SCAMP1-YFP fusions.

ConfocalConfocal共焦的共焦的 immunofluorescenceimmunofluorescence免疫荧光免疫荧光and immunogold electron microscopy studies and immunogold electron microscopy studies demonstrated that YFP-SCAMP1 fusions and demonstrated that YFP-SCAMP1 fusions and native SCAMP1 localize to the plasma membrane native SCAMP1 localize to the plasma membrane and mobile structures in the cytoplasm of and mobile structures in the cytoplasm of transgenic BY-2 cells. transgenic BY-2 cells.

RESULTS RESULTS

1.Highly Conserved Plant 1.Highly Conserved Plant SCAMPsSCAMPs

(A)(A) Alignment of amino Alignment of amino acid sequences of acid sequences of SCAMPs from the rice (SCAMPs from the rice (Oryza sativa) cDNA clone ) cDNA clone used in this study used in this study (SCAMP cDNA); a pea ((SCAMP cDNA); a pea (Pisum sativum) cDNA (Ps ) cDNA (Ps 3941289), an 3941289), an Arabidopsis thalianaArabidopsis thaliana cDNA (At 15220305), cDNA (At 15220305), and an animal (and an animal (Rattus Rattus norvegicusnorvegicus) SCAMP1 ) SCAMP1 cDNA (Rn 3914958).cDNA (Rn 3914958).

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

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

  

2. Generation of Transgenic SCAMP1-YFP BY-2 Cells 2. Generation of Transgenic SCAMP1-YFP BY-2 Cells

and Characterization of SCAMP1 and Characterization of SCAMP1 AntibodiesAntibodies (A)(A) Top panel, chimeric DNA Top panel, chimeric DNA

constructs. YFP was fused at constructs. YFP was fused at either the N terminus of either the N terminus of SCAMP1 (YFP-SCAMP) or the C SCAMP1 (YFP-SCAMP) or the C terminus of SCAMP1 (SCAMP-terminus of SCAMP1 (SCAMP-YFP). The fusions were YFP). The fusions were expressed under the control of expressed under the control of the cauliflower mosaic virus the cauliflower mosaic virus 35S promoter and the 3′ NOS 35S promoter and the 3′ NOS terminator.terminator.

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

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

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

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

3.Subcellular Localization of the SCAMP1-YFP Fusion 3.Subcellular Localization of the SCAMP1-YFP Fusion Construct and SCAMP1 in BY-2 CellsConstruct and SCAMP1 in BY-2 Cells

Subcellular Localization of Subcellular Localization of YFP-SCAMP1 Fusion YFP-SCAMP1 Fusion Constructs in Transgenic Constructs in Transgenic Tobacco BY-2 Cells.Tobacco BY-2 Cells.

Confocal images of YFP Confocal images of YFP signals in cells expressing signals in cells expressing either YFP-SCAMP1 fusion either YFP-SCAMP1 fusion construct were collected construct were collected from untreated cells (panel from untreated cells (panel 1), protoplasts (panel 2), and 1), protoplasts (panel 2), and cells treated with 1.5 M cells treated with 1.5 M NaClNaCl22 (panel 3). (panel 3).

Subcellular Localization of SCAMP1 in Tobacco Subcellular Localization of SCAMP1 in Tobacco BY-2 CellsBY-2 Cells

Panel 1, colocalization Panel 1, colocalization of SCAMP1a and of SCAMP1a and SCAMP1b antibodies in SCAMP1b antibodies in wild-type BY-2 cells; wild-type BY-2 cells;

panel 2, colocalization panel 2, colocalization of SCAMP1-YFP with of SCAMP1-YFP with anti-SCAMP1 in anti-SCAMP1 in transgenic BY-2 cells; transgenic BY-2 cells;

panel 3, colocalization panel 3, colocalization of YFP-SCAMP with of YFP-SCAMP with anti-SCAMP1 in anti-SCAMP1 in transgenic BY-2 cells; transgenic BY-2 cells;