Class 1 reversibly glycosylated polypeptides are plasmodesmal-associated proteins delivered to plasmodesmata via the golgi apparatus - PubMed (original) (raw)
Class 1 reversibly glycosylated polypeptides are plasmodesmal-associated proteins delivered to plasmodesmata via the golgi apparatus
Guy Sagi et al. Plant Cell. 2005 Jun.
Abstract
SE-WAP41, a salt-extractable 41-kD wall-associated protein that is associated with walls of etiolated maize (Zea mays) seedlings and is recognized by an antiserum previously reported to label plasmodesmata and the Golgi, was cloned, sequenced, and found to be a class 1 reversibly glycosylated polypeptide ((C1)RGP). Protein gel blot analysis of cell fractions with an antiserum against recombinant SE-WAP41 showed it to be enriched in the wall fraction. RNA gel blot analysis along the mesocotyl developmental axis and during deetiolation demonstrates that high SE-WAP41 transcript levels correlate spatially and temporally with primary and secondary plasmodesmata (Pd) formation. All four of the Arabidopsis thaliana (C1)RGP proteins, when fused to green fluorescent protein (GFP) and transiently expressed in tobacco (Nicotiana tabacum) epidermal cells, display fluorescence patterns indicating they are Golgi- and plasmodesmal-associated proteins. Localization to the Golgi apparatus was verified by colocalization of transiently expressed AtRGP2 fused to cyan fluorescence protein together with a known Golgi marker, Golgi Nucleotide Sugar Transporter 1 fused to yellow fluorescent protein (GONST1:YFP). In transgenic tobacco, AtRGP2:GFP fluorescence is punctate, is present only in contact walls between cells, and colocalizes with aniline blue-stained callose present around Pd. In plasmolyzed cells, AtRGP2:GFP remains wall embedded, whereas GONST1:YFP cannot be found embedded in cell walls. This result implies that the targeting to Pd is not due to a default pathway for Golgi-localized fusion proteins but is specific to (C1)RGPs. Treatment with the Golgi disrupting drug Brefeldin A inhibits Pd labeling by AtRGP2:GFP. Integrating these data, we conclude that (C1)RGPs are plasmodesmal-associated proteins delivered to plasmodesmata via the Golgi apparatus.
Figures
Figure 1.
Protein Gel Blot Analysis Shows SE-WAP41 Enrichment in Wall Fraction. Proteins extracted from the soluble (s), membranal (m), and wall (w) fractions were separated by SDS-PAGE (6 μg/lane), electroblotted to nitrocellulose, and stained with Ponceau (left), reacted with S-41 antiserum (center), or with SR-41 antiserum (right).
Figure 2.
RNA Gel Slot Blot Analysis of SE-WAP41 mRNA from Mesocotyl Sections of 5-d-Old Etiolated Maize Seedlings. RNA samples were prepared from upper mesocotyl segments: section I contains mesocotyl meristem and region of most rapid cell growth; section II also contains zone of elongation; section III contains mature nondividing and nonelongating cells. The ribosomal 26S rRNA probe served as a loading control. Left, 5-d-old etiolated maize seedling; right, RNA slot blot.
Figure 3.
RNA Gel Blot Analysis Shows That SE-WAP41 mRNA Levels Decrease When Etiolated Maize Seedlings Are Exposed to Light. (A) Five-day-old etiolated maize seedlings were exposed to white light for 3, 6, and 24 h or kept in the dark for 24 h. A blot of total RNA extracted from the upper 1 cm of mesocotyls was hybridized to cDNAs of SE-WAP41, actin, and 26S-rRNA. The ribosomal 26S rRNA probe served as a loading control. The decreased actin mRNA levels is indicative of cell division cessation. (B) Relative SE-WAP41 mRNA levels. Values were calculated relative to 26S-rRNA intensity in each lane.
Figure 4.
SE-WAP41, AtRGP1, AtRGP2, AtRGP3, AtRGP4, and PsRGP1 Share High Amino Acid Sequence Identity. Alignment was made with ClustalX (Thompson et al., 1997). Black boxes denote identical amino acids; gray boxes denote similar amino acids.
Figure 5.
Localization of Transiently Expressed AtRGP2:GFP in Tobacco Epidermal Cells 48 h after Infection by Agrobacterium. (A) Punctate fluorescence can be seen along cell walls as paired fluorescence foci spanning common wall of adjacent cells. (B) A projection of several sections showing AtRGP2:GFP both in fluorescent bodies within cell cytoplasm representing Golgi vesicles and in paired fluorescence foci spanning cell walls. Bars = 10 μm.
Figure 6.
AtRGP2:CFP Colocalizes with the Golgi Apparatus Marker GONST1:YFP. Colocalization is shown in a projection of five optical sections through a tobacco leaf epidermal cell transiently coexpressing AtRGP2:CFP and GONST1:YFP. AtRGP2:CFP fluorescence is shown in (A), GONST1:YFP fluorescence is shown in (B), and both are shown overlaid in (C). Optical sections were taken through the upper cortical cytoplasm near the cuticle where there are no Pd. Fluorescence seen in stomatal guard cells at the right edge of the photograph is autofluorescence induced by the 458-nm irradiation. Bars = 20 μm.
Figure 7.
Stable Expression of AtRGP2:GFP and of MPTMV:GFP in Transgenic Tobacco Plants. Identical fluorescence patterns are presented by leaf epidermal cells in AtRGP2:GFP (A) and MPTMV:GFP (B) expressing transgenic plants: punctate fluorescence spans walls as elongated bars, or paired foci, or appears as single foci inside the cell wall. The areas inside the white boxes are enlarged in the insets in (A) and (B). The trichome-epidermis interface in AtRGP2:GFP (D) and MPTMV:GFP (C) expressing plants displays identical fluorescence patterns. In trichome (F) and spongy mesophyll cells (H) of AtRGP2:GFP expressing transgenic tobacco, fluorescence is detected only in wall areas where there is cell–cell contact and is absent from wall areas without cell–cell contact. To emphasize wall partitions, the same trichome (E) and spongy mesophyll cells (G) are shown with the fluorescence channel turned off. Black arrows indicate wall areas where there is cell–cell contact, and white arrows indicate wall areas without cell–cell contact. Bars = 20 μm.
Figure 8.
AtRGP2:GFP in Transgenic Tobacco Colocalizes with Aniline Blue–Stained Callose Present around Pd. AtRGP2:GFP is shown in green (A), aniline blue–stained callose is shown in blue (B), and both are shown overlaid (C) in a section of cell wall between epidermal cells. Bars = 20 μm.
Figure 9.
Both AtRGP2:GFP and MPTMV:GFP but Not GONST1:YFP Remain inside Cell Walls in Plasmolyzed Cells. The cytoplasmic marker DsRed1 (shown in magenta) was transiently expressed in leaf epidermal cells ∼72 h before plasmolysis. The DsRed1-labeled cytoplasm can be seen to be appressed to cell walls before plasmolysis in AtRGP2:GFP expressing transgenic plants (A) and to retract from walls of plasmolyzed cells in both AtRGP2:GFP expressing transgenic plants (B) and MPTMV:GFP expressing transgenic plants (C). Both GFP fusion proteins (shown in green) remain inside cell walls in areas where the protoplast retracts from junction walls in both AtRGP2:GFP (B) and MPTMV:GFP (C) transgenic plants. By contrast, in GONST1:YFP expressing transgenic plants, GONST1:YFP (shown in yellow) does not remain inside cell walls in areas where the protoplast retracts from junction walls (D). Bars = 20 μm.
Figure 10.
BFA Reduces AtRGP2:GFP Labeling of Both Golgi and Pd. Projections of 10 optical sections through the abaxial side of tobacco leaf epidermal cells treated with BFA (A) or mock treated (B). Bars = 20 μm.
Figure 11.
An Illustration Showing That the Nature of a Protein's Association with Golgi Determines Its Destination inside a Pd. (A) Proteins residing in the Golgi lumen are exported to the cell wall surrounding the Pd. (B) Proteins with transmembrane regions arrive at the PM delineating the Pd. (C) Proteins peripherally associated with the cytosolic side of the Golgi membrane reach the inner side of the PM facing the cytoplasmic sleeve of the Pd.
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