N-Glycomic and Microscopic Subcellular Localization Analyses of NPP1, 2 and 6 Strongly Indicate thattrans-Golgi Compartments Participate in the Golgi to Plastid Traffic of Nucleotide Pyrophosphatase/Phosphodiesterases in Rice

Abstract

Nucleotide pyrophosphatase/phosphodiesterases (NPPs) are widely distributed N-glycosylated enzymes that catalyze the hydrolytic breakdown of numerous nucleotides and nucleotide sugars. In many plant species, NPPs are encoded by a small multigene family, which in rice are referred to NPP1–NPP6. Although recent investigations showed that N-glycosylated NPP1 is transported from the endoplasmic reticulum (ER)–Golgi system to the chloroplast through the secretory pathway in rice cells, information on N-glycan composition and subcellular localization of other NPPs is still lacking. Computer-assisted analyses of the amino acid sequences deduced from different Oryza sativa NPP-encoding cDNAs predicted all NPPs to be secretory glycoproteins. Confocal fluorescence microscopy observation of cells expressing NPP2 and NPP6 fused with green fluorescent protein (GFP) revealed that NPP2 and NPP6 are plastidial proteins. Plastid targeting of NPP2–GFP and NPP6–GFP was prevented by brefeldin A and by the expression of ARF1(Q71L), a dominant negative mutant of ADP-ribosylation factor 1 that arrests the ER to Golgi traffic, indicating that NPP2 and NPP6 are transported from the ER–Golgi to the plastidial compartment. Confocal laser scanning microscopy and high-pressure frozen/freeze-substituted electron microscopy analyses of transgenic rice cells ectopically expressing the trans-Golgi marker sialyltransferase fused with GFP showed the occurrence of contact of Golgi-derived membrane vesicles with cargo and subsequent absorption into plastids. Sensitive and high-throughput glycoblotting/mass spectrometric analyses showed that complex-type and paucimannosidic-type glycans with fucose and xylose residues occupy approximately 80% of total glycans of NPP1, NPP2 and NPP6. The overall data strongly indicate that the trans-Golgi compartments participate in the Golgi to plastid trafficking and targeting mechanism of NPPs.

Altered expression of polyamine transporters reveals a role for spermidine in the timing of flowering and other developmental response pathways

“…The dual targeting of this protein was observed in WT plants, transiently expressed protoplasts of A. thaliana and agroinfiltrated tobacco leaves. Some proteins such as the rice alpha amylase protein, and nucleotide pyrophosphatase/phosphodiesterases [32] are targeted to the plastid after first passing through the Golgi [33]. Protoplasts have been a valuable resource for localization studies, but protoplasts prepared from leaf tissues are not identical cell types, and the localization of some proteins is both tissue and cell-type dependent [34].…”

Lipid transport required to make lipids of photosynthetic membranes

“…Evidence for secretory (Im et al 2005;Saravanan et al 2009) c Ref: (Shaw 1996;Stefan et al 2011;Watt et al 2002) d Ref: (Davletov and Sudhof 1993;Pérez-Sancho et al 2015) vesicles fusing with the chloroplast envelopes comes from the transport of fully glycoslyated proteins to the chloroplast stroma (Kitajima et al 2009;Nanjo et al 2006;Villarejo et al 2005). A subset of these proteins require glycosylation for function, further evidence of their transport through the secretory pathway (Buren et al 2011;Kaneko et al 2016). Although it seems likely that the proteins are transported through vesicles, the mechanism by which vesicles fuse with the chloroplast is unknown (Baslam et al 2016).…”

“…Baslam M, Oikawa K, Kitajima-Koga A,Kaneko K, Mitsui T (2016) Golgi-to-plastid trafficking of proteins through secretory pathway: Insights into vesiclemediated import toward the plastids. PlantSignaling & Behavior 11 doi:10.108 0/15592324.2016.1221558 Bates PD, Ohlrogge JB, Pollard M (2007 Incorporation of newly synthesized fatty acids into cytosolic glycerolipids in pea leaves occurs via acyl editing.…”

Golgi-to-plastid trafficking of proteins through secretory pathway: Insights into vesicle-mediated import toward the plastids

“… 4 This localization pattern was further supported using onion epidermal cells transiently expressing NPP-GFP. 4 It is worth noting that the remaining (18–27%) background signal was distributed among the ER, Golgi apparatus and/or the other endomembrane systems. These results strongly suggest that NPP glycoproteins localize in the plastid.…”