Download Rice SCAMP1 Defines Clathrin-Coated, trans

Rice SCAMP1 Defines
Clathrin-Coated, transGolgi–Located TubularVesicular 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.

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.

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 3941289), an
Arabidopsis thaliana
cDNA (At 15220305),
and an animal (Rattus
norvegicus) SCAMP1
cDNA (Rn 3914958).

Gray areas indicate
conserved amino acids
among the four SCAMPs.
Two synthetic peptides,
corresponding to the Nterminal 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 Cterminal 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 (SCAMPYFP). 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 SDSPAGE 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 wildtype 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 antiSCAMP1b. 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.
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.
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
NaCl2 (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 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;