Arabinogalactan glycosyltransferases target to a unique subcellular compartment that may function in unconventional secretion in plants.
Bottom Line: Approximately 80% of AtGALT31A [Arabidopsis thaliana galactosyltransferase from family 31 (At1g32930)] was found in the small compartments, of which, 45 and 40% of AtGALT29A [Arabidopsis thaliana galactosyltransferase from family 29 (At1g08280)] and AtGlcAT14A [Arabidopsis thaliana glucuronosyltransferase from family 14 (At5g39990)] colocalized with AtGALT31A, respectively; in contrast, N-glycosylation enzymes rarely colocalized (3-18%), implicating a role of the small compartments in a part of arabinogalactan (O-glycan) biosynthesis rather than N-glycan processing.The AtGALT31A localized to the small compartments were colocalized with neither SYP61 (syntaxin of plants 61), a marker for trans-Golgi network (TGN), nor FM4-64-stained endosomes.Taken together, AtGALT31A localized to small compartments that are distinct from the Golgi apparatus, the SYP61-localized TGN, FM4-64-stained endosomes and Wortmannin-vacuolated prevacuolar compartments, but may be part of an unconventional protein secretory pathway represented by EXO70E2 in plants.
Affiliation: Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Thorvaldsensvej 40, Frederiksberg C, 1871, Denmark.Show MeSH
Mentions: We characterized the AtGALT31A-localized small compartments further using chemicals, such as FM4-64, BFA and Wortmannin. FM4-64 is used as an endocytic tracer that internalizes and stains early endosomes 25–28. We investigated the colocalization of the AtGALT31A-localized small compartments and the early endosome stained by the internalized FM4-64 25–28 and compared with the SYP61-localized TGN. In the N. benthamiana leaves used in this study, we observed a clear internalization of FM4-64, which demonstrated a sequential staining from the plasma membrane, internal compartments, and aggregation of the compartments over time (Figure 8). In this condition, the AtGALT31A-localized small compartments were not colocalized with the FM4-64-stained compartments for up to 140 min (Figure 8A–C). We had intended to use SYP61 as a positive marker for the colocalization with the FM4-64-stained early endosomes 29–32, however, we observed that SYP61-containing TGN subdomains did not colocalize with FM4-64-stained compartments for up to 140 min (Figure 8D–F). The reason for this discrepancy with the previous reports remains unknown. However, FM4-64 clearly worked properly in our conditions; therefore, this difference may be due to the definition of TGN domains by different markers. Most of the published studies for TGN have been performed using VHA-a1 as a marker, but the distinct properties of the TGN subdomains as defined by VHA-a1 or SYP61 expressed in N. benthamiana leaves have been previously reported 33. Additionally, the TGN in N. benthamiana leaves characterized in this study may have slightly different properties than the TGN in the Arabidopsis root used in most of the prior publications. This variation can be implicated by the different properties of the Golgi apparatus and the TGN in response to BFA and its analogue from different plant species and tissues as reported in 34. Nevertheless, under the experimental conditions used in this article, the AtGALT31A-localized small compartments did not colocalize with the FM4-64-stained compartments for up to 140 min (Figure 8A–C), indicating an exclusion of the AtGALT31A-localized small compartments from the endocytic pathway followed by FM4-64.
Affiliation: Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Thorvaldsensvej 40, Frederiksberg C, 1871, Denmark.