Limits...
Two novel types of hexokinases in the moss Physcomitrella patens.

Nilsson A, Olsson T, Ulfstedt M, Thelander M, Ronne H - BMC Plant Biol. (2011)

Bottom Line: However, we also found two new types of hexokinases with no obvious orthologs in vascular plants.Type C, encoded by a single gene, has neither transit peptide nor membrane anchor, and is found in the cytosol and in the nucleus.We conclude that the hexokinase gene family is more diverse in Physcomitrella, encoding two additional types of hexokinases that are absent in vascular plants.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology, Swedish University of Agricultural Sciences, Box 7025, SE-750 07 Uppsala, Sweden.

ABSTRACT

Background: Hexokinase catalyzes the phosphorylation of glucose and fructose, but it is also involved in sugar sensing in both fungi and plants. We have previously described two types of hexokinases in the moss Physcomitrella. Type A, exemplified by PpHxk1, the major hexokinase in Physcomitrella, is a soluble protein that localizes to the chloroplast stroma. Type B, exemplified by PpHxk2, has an N-terminal membrane anchor. Both types are found also in vascular plants, and localize to the chloroplast stroma and mitochondrial membranes, respectively.

Results: We have now characterized all 11 hexokinase encoding genes in Physcomitrella. Based on their N-terminal sequences and intracellular localizations, three of the encoded proteins are type A hexokinases and four are type B hexokinases. One of the type B hexokinases has a splice variant without a membrane anchor, that localizes to the cytosol and the nucleus. However, we also found two new types of hexokinases with no obvious orthologs in vascular plants. Type C, encoded by a single gene, has neither transit peptide nor membrane anchor, and is found in the cytosol and in the nucleus. Type D hexokinases, encoded by three genes, have membrane anchors and localize to mitochondrial membranes, but their sequences differ from those of the type B hexokinases. Interestingly, all moss hexokinases are more similar to each other in overall sequence than to hexokinases from other plants, even though characteristic sequence motifs such as the membrane anchor of the type B hexokinases are highly conserved between moss and vascular plants, indicating a common origin for hexokinases of the same type.

Conclusions: We conclude that the hexokinase gene family is more diverse in Physcomitrella, encoding two additional types of hexokinases that are absent in vascular plants. In particular, the presence of a cytosolic and nuclear hexokinase (type C) sets Physcomitrella apart from vascular plants, and instead resembles yeast, where all hexokinases localize to the cytosol. The fact that all moss hexokinases are more similar to each other than to hexokinases from vascular plants, even though both type A and type B hexokinases are present in all plants, further suggests that the hexokinase gene family in Physcomitrella has undergone concerted evolution.

Show MeSH
Intracellular localization of Physcomitrella hexokinase-GFP fusions. Fluorescence microscopy pictures of wild type protoplasts transiently expressing different GFP fusions. GFP fluorescence is shown in green, with the chlorophyll auto-fluorescence in red as a chloroplast marker. Protoplasts expressing GFP alone were also included as a control. The white bars represent 5 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3045890&req=5

Figure 4: Intracellular localization of Physcomitrella hexokinase-GFP fusions. Fluorescence microscopy pictures of wild type protoplasts transiently expressing different GFP fusions. GFP fluorescence is shown in green, with the chlorophyll auto-fluorescence in red as a chloroplast marker. Protoplasts expressing GFP alone were also included as a control. The white bars represent 5 μm.

Mentions: We proceeded to study the intracellular locations of the moss hexokinases. Sequences from the new hexokinases, expressed from the 35S promoter, were fused in frame to GFP. These constructs were transiently expressed in Physcomitrella protoplasts and the GFP fluorescence was monitored (Figure 4). Based on the sequence similarity of the N-terminal membrane anchors in PpHxk2, PpHxk3, PpHxk7 and PpHxk8 to those found in AtHxk2 (Figure 2) we expected that they would localize to the outer mitochondrial membrane, as shown for AtHxk2 and several other type B hexokinases [7-9,12,13,49]. Consistent with this, we found that the Physcomitrella type B hexokinases tested also localize to small ring-like membrane structures (Figure 4) which were identified as mitochondrial membranes by co-staining with MitoTracker® (Figure 5). In contrast, truncated GFP fusions which lacked the membrane anchors showed a diffuse localization throughout the cell (Additional file 6: Figure S2). We conclude that the N-terminal membrane anchors target the proteins to the mitochondria. We further note that the mitochondria often formed aggregates (Figure 5). This may be an artefact caused by protein overexpression, as shown for other membrane-anchored GFP fusions expressed in plants [50]. A similar aggregation of mitochondria was also seen when several of the Arabidopsis hexokinase GFP fusions were overexpressed [13].


Two novel types of hexokinases in the moss Physcomitrella patens.

Nilsson A, Olsson T, Ulfstedt M, Thelander M, Ronne H - BMC Plant Biol. (2011)

Intracellular localization of Physcomitrella hexokinase-GFP fusions. Fluorescence microscopy pictures of wild type protoplasts transiently expressing different GFP fusions. GFP fluorescence is shown in green, with the chlorophyll auto-fluorescence in red as a chloroplast marker. Protoplasts expressing GFP alone were also included as a control. The white bars represent 5 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3045890&req=5

Figure 4: Intracellular localization of Physcomitrella hexokinase-GFP fusions. Fluorescence microscopy pictures of wild type protoplasts transiently expressing different GFP fusions. GFP fluorescence is shown in green, with the chlorophyll auto-fluorescence in red as a chloroplast marker. Protoplasts expressing GFP alone were also included as a control. The white bars represent 5 μm.
Mentions: We proceeded to study the intracellular locations of the moss hexokinases. Sequences from the new hexokinases, expressed from the 35S promoter, were fused in frame to GFP. These constructs were transiently expressed in Physcomitrella protoplasts and the GFP fluorescence was monitored (Figure 4). Based on the sequence similarity of the N-terminal membrane anchors in PpHxk2, PpHxk3, PpHxk7 and PpHxk8 to those found in AtHxk2 (Figure 2) we expected that they would localize to the outer mitochondrial membrane, as shown for AtHxk2 and several other type B hexokinases [7-9,12,13,49]. Consistent with this, we found that the Physcomitrella type B hexokinases tested also localize to small ring-like membrane structures (Figure 4) which were identified as mitochondrial membranes by co-staining with MitoTracker® (Figure 5). In contrast, truncated GFP fusions which lacked the membrane anchors showed a diffuse localization throughout the cell (Additional file 6: Figure S2). We conclude that the N-terminal membrane anchors target the proteins to the mitochondria. We further note that the mitochondria often formed aggregates (Figure 5). This may be an artefact caused by protein overexpression, as shown for other membrane-anchored GFP fusions expressed in plants [50]. A similar aggregation of mitochondria was also seen when several of the Arabidopsis hexokinase GFP fusions were overexpressed [13].

Bottom Line: However, we also found two new types of hexokinases with no obvious orthologs in vascular plants.Type C, encoded by a single gene, has neither transit peptide nor membrane anchor, and is found in the cytosol and in the nucleus.We conclude that the hexokinase gene family is more diverse in Physcomitrella, encoding two additional types of hexokinases that are absent in vascular plants.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology, Swedish University of Agricultural Sciences, Box 7025, SE-750 07 Uppsala, Sweden.

ABSTRACT

Background: Hexokinase catalyzes the phosphorylation of glucose and fructose, but it is also involved in sugar sensing in both fungi and plants. We have previously described two types of hexokinases in the moss Physcomitrella. Type A, exemplified by PpHxk1, the major hexokinase in Physcomitrella, is a soluble protein that localizes to the chloroplast stroma. Type B, exemplified by PpHxk2, has an N-terminal membrane anchor. Both types are found also in vascular plants, and localize to the chloroplast stroma and mitochondrial membranes, respectively.

Results: We have now characterized all 11 hexokinase encoding genes in Physcomitrella. Based on their N-terminal sequences and intracellular localizations, three of the encoded proteins are type A hexokinases and four are type B hexokinases. One of the type B hexokinases has a splice variant without a membrane anchor, that localizes to the cytosol and the nucleus. However, we also found two new types of hexokinases with no obvious orthologs in vascular plants. Type C, encoded by a single gene, has neither transit peptide nor membrane anchor, and is found in the cytosol and in the nucleus. Type D hexokinases, encoded by three genes, have membrane anchors and localize to mitochondrial membranes, but their sequences differ from those of the type B hexokinases. Interestingly, all moss hexokinases are more similar to each other in overall sequence than to hexokinases from other plants, even though characteristic sequence motifs such as the membrane anchor of the type B hexokinases are highly conserved between moss and vascular plants, indicating a common origin for hexokinases of the same type.

Conclusions: We conclude that the hexokinase gene family is more diverse in Physcomitrella, encoding two additional types of hexokinases that are absent in vascular plants. In particular, the presence of a cytosolic and nuclear hexokinase (type C) sets Physcomitrella apart from vascular plants, and instead resembles yeast, where all hexokinases localize to the cytosol. The fact that all moss hexokinases are more similar to each other than to hexokinases from vascular plants, even though both type A and type B hexokinases are present in all plants, further suggests that the hexokinase gene family in Physcomitrella has undergone concerted evolution.

Show MeSH