Characterisation of Arabidopsis calnexin 1 and calnexin 2 in the endoplasmic reticulum and at plasmodesmata

Abstract

Calnexin (CNX) is a highly conserved endoplasmic reticulum (ER) chaperone protein. Both calnexin and the homologous ER-lumenal protein, calreticulin, bind calcium ions and participate in protein folding. There are two calnexins in Arabidopsis thaliana, CNX1 and CNX2. GUS expression demonstrated that these are expressed in most Arabidopsis tissues throughout development. Calnexin transfer DNA (T-DNA) mutant lines exhibited increased transcript abundances of a number of other ER chaperones, including calreticulins, suggesting a degree of redundancy. CNX1 and CNX2 localised to the ER membrane including that within plasmodesmata, the intercellular channels connecting plant cells. This is comparable with the previous localisations of calreticulin in the ER lumen and at plasmodesmata. However, from green fluorescent protein (GFP) diffusion studies in single and double T-DNA insertion mutant lines, as well as overexpression lines, we found no evidence that CNX1 or CNX2 play a role in intercellular transport through plasmodesmata. In addition, calnexin T-DNA mutant lines showed no change in transcript abundance of a number of plasmodesmata-related proteins. CNX1 and CNX2 do not appear to have a specific localisation or function at plasmodesmata-rather the association of calnexin with the ER is simply maintained as the ER passes through plasmodesmata.

A Split-GFP Gateway Cloning System for Topology Analyses of Membrane Proteins in Plants

“…The same was true when these two moieties were co-expressed on the luminal side of the ER membrane, despite the different redox and ionic conditions in this compartment. This was in agreement with previous use of split-GFP in mammalian cells and the known topology of calnexin [7,15]. …”

“…To subsequently test this membrane protein topology analysis system, we selected to use the transmembrane domain (CNX TM ) of the Arabidopsis ER protein, calnexin 1. Calnexins are ER-membrane integral proteins with an N-terminal Ca 2+ -binding chaperone domain in the ER lumen and a C-terminal transmembrane domain [14,15]. Only when CNX TM -GFP11 was co-expressed with cytosolic GFP1-10 and when GFP11-CNX TM was co-expressed with ER-luminal GFP1-10, did we observe strong GFP signal at the ER cortical network in Nicotiana benthamiana leaf epidermal cells.…”

Basic‐leucine zipper 17 and Hmg‐CoA reductase degradation 3A are involved in salt acclimation memory in Arabidopsis

“…Proteins involved in these activities are usually located in the ER (Helenius and Aebi 2004;Howell 2013;Christianson and Ye 2014). Indeed, many proteins encoded by genes in Group C were predicted to be localized to the ER lumen and ER membrane based on GOseq analysis (Young et al 2010), such as CNX2, DER2.1, AT2G21190, AT3G15710, and AT3G17780 (Table S5) (Kamauchi et al 2005;Liu et al 2015), suggesting the salt acclimation memory may be closely related to the ER.…”

Membranes of unification

“…The role of the ER as intercellular connector is addressed by Liu et al (2016) in the current issue. In this work, the senior author returns to a seminal paper published more than three decades ago, where the continuity of the ER with the desmotubule had been shown (Overall et al 1982).…”

“…In this work, the senior author returns to a seminal paper published more than three decades ago, where the continuity of the ER with the desmotubule had been shown (Overall et al 1982). In the current work, they look at localisation and function of calnexins, which, together with their homologue calreticulin, are major calcium-binding proteins in the ER involved in protein folding.…”