Multiple N-Glycans Cooperate in the Subcellular Targeting and Functioning of Arabidopsis KORRIGAN1

Abstract

Arabidopsis thaliana KORRIGAN1 (KOR1) is an integral membrane endo-b1,4-glucanase in the trans-Golgi network and plasma membrane that is essential for cellulose biosynthesis. The extracellular domain of KOR1 contains eight N-glycosylation sites, N1 to N8, of which only N3 to N7 are highly conserved. Genetic evidence indicated that cellular defects in attachment and maturation of these N-glycans affect KOR1 function in vivo, whereas the manner by which N-glycans modulate KOR1 function remained obscure. Site-directed mutagenesis analysis of green fluorescent protein (GFP)-KOR1 expressed from its native regulatory sequences established that all eight N-glycosylation sites (N1 to N8) are used in the wild type, whereas stt3a-2 cells could only inefficiently add N-glycans to less conserved sites. GFP-KOR1 variants with a single N-glycan at nonconserved sites were less effective than those with one at a highly conserved site in rescuing the root growth phenotype of rsw2-1 (kor1 allele). When functionally compromised, GFP-KOR1 tended to accumulate at the tonoplast. GFP-KOR1Dall (without any N-glycan) exhibited partial complementation of rsw2-1; however, root growth of this line was still negatively affected by the absence of complex-type N-glycan modifications in the host plants. These results suggest that one or several additional factor(s) carrying complex N-glycans cooperate(s) with KOR1 in trans to grant proper targeting/functioning in plant cells.

Arabidopsis thalianaKORRIGAN1 protein: N-glycan modification, localization, and function in cellulose biosynthesis and osmotic stress responses

“…4 This was particularly evident for less conserved sites, i.e., N-glycosylation at N1 or N2 were undetectable, and N8 showed approximately 50% occupation. In the salt-sensitve stt3a-2 mutant, GFP-KOR1 predominantly accumulated at the tonoplast with some retention in the TGN, which was much more severe than expected from the moderate underglycosylation of KOR1 at 3 to 4 sites, suggesting that additional (glyco)proteins might function in retaining KOR1 within the TGN.…”

“…3 GHF9 endoglucanases are widespread in both Prokaryota and Eukaryota including all plant cellulases. The KOR1 sequence contains 8 N-glycosylation motifs (N1-N8), of which 5 are highly conserved among plant GHF9 proteins, 4 suggesting that N-glycosylation of KOR1 may be essential for function. Our genetic interaction analyses detected strong synergistic effects between KOR1 partial loss-of-function allele rsw2-1 and other mutations defective in either N-glycan attachment (stt3a-2) in the endoplasmic reticulum (ER) or complex N-glycan maturation in the Golgi apparatus (cgl1-3).…”

“…Fourth, even non-glycosylated KOR1 can partially complement rsw2-1, suggesting that KOR1 without Nglycans retains some activity, or KOR1 may have non-catalytic functions as suggested previously. 3,4 In wild-type plants, intact GFP-KOR1 localized predominately in the trans-Golgi network (TGN), the plasma membrane (PM), and at low levels also in tonoplasts. 4 Cycling of KOR1 between the PM and heterogeneous intracellular compartments has been reported.…”

“…3,4 In wild-type plants, intact GFP-KOR1 localized predominately in the trans-Golgi network (TGN), the plasma membrane (PM), and at low levels also in tonoplasts. 4 Cycling of KOR1 between the PM and heterogeneous intracellular compartments has been reported. 10 During cell division, KOR1 becomes enriched at the cell plate, which is promoted by a polarized targeting signal (LL and YXXF) in the cytoplasmic domain.…”

“…To determine the in-vivo function of KOR1 N-glycans, a systematic mutagenesis of all 8 N-glycosylation sites was conducted in our recent study. 4 Functional complementation using GFP-KOR1 variants, whose expression was driven by native KOR1 promoter and terminator sequences, were analyzed in KOR1-deficient mutant host rsw2-1, and the N-glycosylation state of KOR1 and the root-growth phenotype of the transformants were scored. From these analyses several conclusions could be drawn.…”