Abstract
SummaryFasciclin‐like arabinogalactan proteins (FLAs) are involved in numerous important functions in plants but the relevance of their complex structure to physiological function and cellular fate is unresolved. Using a fully functional fluorescent version of Arabidopsis thaliana FLA4 we show that this protein is localized at the plasma membrane as well as in endosomes and soluble in the apoplast. FLA4 is likely to be GPI‐anchored, is highly N‐glycosylated and carries two O‐glycan epitopes previously associated with arabinogalactan proteins. The activity of FLA4 was resistant against deletion of the amino‐proximal fasciclin 1 domain and was unaffected by removal of the GPI‐modification signal, a highly conserved N‐glycan or the deletion of predicted O‐glycosylation sites. Nonetheless these structural changes dramatically decreased endoplasmic reticulum (ER)‐exit and plasma membrane localization of FLA4, with N‐glycosylation acting at the level of ER‐exit and O‐glycosylation influencing post‐secretory fate. We show that FLA4 acts predominantly by molecular interactions involving its carboxy‐proximal fasciclin 1 domain and that its amino‐proximal fasciclin 1 domain is required for stabilization of plasma membrane localization. FLA4 functions as a soluble glycoprotein via its carboxy‐proximal Fas1 domain and its normal cellular trafficking depends on N‐ and O‐glycosylation.
Highlights
Arabinogalactan proteins (AGPs) are a large protein family in plants implicated with many biological functions (Seifert and Roberts, 2007; Ellis et al, 2010)
After fasciclin I was identified in axon fascicles of insects (Bastiani et al, 1987; Snow et al, 1988; Zinn et al, 1988), proteins carrying the fasciclin 1 (Fas1) domain were found in animals, plants, fungi, eubacteria and archaea, and were suggested to act at the interface between the extracellular matrix (ECM) and intracellular signalling (Elkins et al, 1990; Thapa et al, 2007; Harris and Weigel, 2008; Kim et al, 2012; Ghatak et al, 2014)
We generated FLA4-citrin (F4C), an in-frame fusion between the predicted FLA4 signal peptide fused to the pH-stable yellow fluorescent protein monomeric citrin (Shaner et al, 2005) followed by the FLA4 coding region including the predicted GPI-signal (Figure S1), driven by various promoters and transformed sos5 mutant plants with the constructs
Summary
Arabinogalactan proteins (AGPs) are a large protein family in plants implicated with many biological functions (Seifert and Roberts, 2007; Ellis et al, 2010). After fasciclin I was identified in axon fascicles of insects (Bastiani et al, 1987; Snow et al, 1988; Zinn et al, 1988), proteins carrying the fasciclin 1 (Fas1) domain were found in animals, plants, fungi, eubacteria and archaea, and were suggested to act at the interface between the extracellular matrix (ECM) and intracellular signalling (Elkins et al, 1990; Thapa et al, 2007; Harris and Weigel, 2008; Kim et al, 2012; Ghatak et al, 2014). The four human Fas proteins function in cell to ECM adhesion, as ECM structural elements, in glucosaminoglycan serum clearance, as paracrine signals and in signal perception and they interact with ECM receptors of the integrin family (Kim et al, 2000; Harris et al, 2008; Zhou et al, 2015; Bonnet et al, 2016). The function of Fas proteins in plants is exclusively understood at the genetic
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