The cellulose‐deficient Arabidopsis mutant rsw3 is defective in a gene encoding a putative glucosidase II, an enzyme processing N‐glycans during ER quality control

Abstract

Summaryrsw3 is a temperature-sensitive mutant of Arabidopsis thaliana showing radially swollen roots and a deficiency in cellulose. The rsw3 gene was identified by a map-based strategy, and shows high similarity to the catalytic a-subunits of glucosidase II from mouse, yeast and potato. These enzymes process N-linked glycans in the ER, so that they bind and then release chaperones as part of the quality control pathway, ensuring correct protein folding. Putative b-subunits for the glucosidase II holoenzyme identified in the Arabidopsis and rice genomes share characteristic motifs (including an HDEL ER-retention signal) with bsubunits in mammals and yeast. The genes encoding the putative a-and b-subunits are single copy and, like the rsw3 phenotype, widely expressed. rsw3 reduces cell number more strongly than cell size in stamen filaments and probably stems. Most features of the rsw3 phenotype are shared with other cellulose-deficient mutants, but some -notably, production of multiple rosettes and a lack of secreted seed mucilageare not and may reflect glucosidase II affecting processes other than cellulose synthesis. The rsw3 root phenotype develops more slowly than the rsw1 and rsw2 phenotypes when seedlings are transferred to the restrictive temperature. This is consistent with rsw3 reducing glycoprotein delivery from the ER to the plasma membrane whereas rsw1 and rsw2 act more rapidly by affecting the properties of already delivered enzymes.

The STT3a Subunit Isoform of the Arabidopsis Oligosaccharyltransferase Controls Adaptive Responses to Salt/Osmotic Stress

“…This phenotype is unlike that of sos1 seedlings, for which salt/osmotic stress sensitivity is associated with the death of root meristematic cells (Huh et al, 2002). Instead, the salt/osmotic stress-responsive phenotype of stt3a seedlings resembles the phenotype caused by the mutation to the RSW3 locus ( rsw3-1 ) that encodes ␣ -glucosidase II (Burn et al, 2002). At restrictive temperatures, rsw3-1 causes the inhibition of cell division (Burn et al, 2002).…”

“…Instead, the salt/osmotic stress-responsive phenotype of stt3a seedlings resembles the phenotype caused by the mutation to the RSW3 locus ( rsw3-1 ) that encodes ␣ -glucosidase II (Burn et al, 2002). At restrictive temperatures, rsw3-1 causes the inhibition of cell division (Burn et al, 2002). To determine whether salt/osmotic stress inhibits root cell mitotic activity in stt3a seedlings, the expression of CycB1;1 was monitored ( Figure 4).…”

α-Mannosidase Involved in Turnover of Plant Complex TypeN-Glycans in Tomato (Lycopersicum esculentum) Fruits

“…2,3) The former -mannosidases reside in the endoplasmic reticulum (ER) or Golgi apparatus and play a critical role in the early stage of processing pathway of N-glycans. 1) In plant cells, it has been found that N-glycan processing by -glucosidase I and II in the ER is critical to growth and development, [4][5][6] but N-glycan processing in Golgi apparatus appeared to have little effect on normal growth. Arabidopsis mutants that lack N-acetylglucosaminyltransferase I (GNT-I) 7) or bothxylosyltransferase ( 1-2XylT) and -fucosyltransferase ( 1-3FucT) 8) grow normally and produce normal seeds.…”

N‐glycomic profiling of a glucosidase II mutant of Dictyostelium discoideum by ‘‘off‐line’’ liquid chromatography and mass spectrometry

“…RT-PCR using mRNA from the M31 strain was performed using primers specific for the modA gene and the fragments were ligated into a standard cloning vector; sequencing of clones derived from two independent PCR reactions showed the same G→A mutation at nucleotide 1931 of the open reading frame which would result in a substitution of a conserved glycine residue by aspartate (Gly644→Asp; Figure 1). This amino acid is in a region associated with glucosidase II mutations in other organisms -in particular, the Ser599→Phe alteration in the Arabidopsis rsw3 mutant (9) and a frameshift in the Trichoderma Rut-30C strain (13) . We propose that this alteration results in a diminution or abolition of glucosidase II activity, which in turn has an impact on the N-glycome of Dictyostelium; indeed, in an initial screen for glycomic differences in M31, Western blotting with anti-horseradish peroxidase (anti-HRP), wheat germ agglutinin (WGA) and a single-chain antibody specific for mannose-6-phosphate showed, respectively, that core α1,3-fucosylation is absent, that WGA reactivity (possibly to intersecting and bisecting GlcNAc) is reduced and that mannose-6-phosphate is still present (data not shown).…”

“…The biological role of glucosidase II (EC 3.2.1.84) has been assessed with a range of mutant cell lines and organisms, including Arabidopsis thaliana, Candida albicans, Saccharomyces cerevisiae and Trypanosoma brucei knock-outs as well as a hypercellulolytic strain of Trichoderma reesei, a mouse lymphoma cell line and the Dicytostelium discoideum modA (M31) mutant (9)(10)(11)(12)(13)(14)(15) . A recurring feature of the plant and fungal mutants is the occurrence of cell wall phenotypes suggesting that glucosidase II is required for proper formation of cell walls; indeed in Dictyostelium, which also produces cellulose, stunted fruiting bodies are the obvious morphological phenotype of the modA strain.…”