Class 1 Reversibly Glycosylated Polypeptides Are Plasmodesmal-Associated Proteins Delivered to Plasmodesmata via the Golgi Apparatus

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 (C1RGP). 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 C1RGP 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 C1RGPs. Treatment with the Golgi disrupting drug Brefeldin A inhibits Pd labeling by AtRGP2:GFP. Integrating these data, we conclude that C1RGPs are plasmodesmal-associated proteins delivered to plasmodesmata via the Golgi apparatus.

AnArabidopsisGPI-Anchor Plasmodesmal Neck Protein with Callose Binding Activity and Potential to Regulate Cell-to-Cell Trafficking

“…By analogy with the nuclear pore complex, also mediating macromolecular trafficking, we might expect there to be many tens of proteins involved in Pd structure and function. Recent successes in identifying Pd proteins indicate that the identification of the constituent components of Pds need not remain the major hurdle in understanding macromolecular trafficking in plants ( Lee et al, 2005;Sagi et al, 2005;Levy et al, 2007b;Thomas et al, 2008). However, the proteins identified so far show few examples of functional connectivity, indicating that many plasmodesmal proteins remain to be identified.…”

“…Various approaches have been taken to identify such constituents, with limited success (reviewed in Oparka, 2004;Maule, 2008). The proteins identified include cytoskeletal elements (e.g., actin and myosin VIII) (Ding et al, 1996;Blackman and Overall, 1998;Reichelt et al, 1999), proteins associated with the endoplasmic reticulum (e.g., calreticulin) , a protein kinase 1 that phosphorylates Tobacco mosaic virus movement protein (Lee et al, 2005), a 1,3-b-glucanase (Levy et al, 2007b), a class 1 reversibly glycosylated protein (Sagi et al, 2005), and, recently, a receptor-like transmembrane protein located on the plasma membrane in Pds (Thomas et al, 2008). In this article, we report the identification of a family of small glycosylphosphatidylinositol (GPI)-linked proteins that exhibit specific callose binding activity in vitro and that localize to Pds as fluorescent protein fusions.…”

Ultrastructural study of plasmodesmata in the brown alga Dictyota dichotoma (Dictyotales, Phaeophyceae)

“…Several other works noted additional internal substructures (Ding et al 1992b, Overall and Blackman 1996. Recently, PD-associated proteins have been identified in charophycean algae and land plants , Blackman and Overall 1998, Gestel et al 2003, Faulkner et al 2005, Sagi et al 2005, Thomas et al 2008, Simpson et al 2009, Faulkner and Maule 2010. These works suggested that there are highly complicated control mechanisms underlying the cell-to-cell molecular traffic via PD.…”

Grain setting defect1, Encoding a Remorin Protein, Affects the Grain Setting in Rice through Regulating Plasmodesmatal Conductance

“…PD are transverse cell wall channels structured with the cytoplasmic sleeve and the modified endoplasmic reticulum desmotubule between neighboring cells (Maule, 2008). A number of proteins affect the structure and functional performance of the PD, which in turn impacts the cell-to-cell transport of small and large molecules through the PD during plant growth, development, and defense (Cilia and Jackson, 2004;Sagi et al, 2005;Lucas et al, 2009;Simpson et al, 2009;Stonebloom et al, 2009). For example, actin and myosin, which link the desmotubule to the plasma membrane (PM) at the neck region of PD, are believed to play a role in regulating PD permeability by controlling PD aperture (White et al, 1994;Ding et al, 1996;Reichelt et al, 1999).…”