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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.

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PpHxk3 can complement a hexokinase-deficient yeast strain. The picture shows growth of the hxk1 hxk2 glk1 triple disrupted yeast strain containing the pFL61 vector with different inserts on plates containing 2% galactose, 2% glucose or 3% raffinose as carbon source. Growth on glucose requires glucokinase activity and growth on raffinose fructokinase activity. The inserts from left to right are: PpHXK3 cDNA; PpHXK1 cDNA encoding an N-terminally truncated protein; PpHXK1 cDNA; no insert.
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Figure 8: PpHxk3 can complement a hexokinase-deficient yeast strain. The picture shows growth of the hxk1 hxk2 glk1 triple disrupted yeast strain containing the pFL61 vector with different inserts on plates containing 2% galactose, 2% glucose or 3% raffinose as carbon source. Growth on glucose requires glucokinase activity and growth on raffinose fructokinase activity. The inserts from left to right are: PpHXK3 cDNA; PpHXK1 cDNA encoding an N-terminally truncated protein; PpHXK1 cDNA; no insert.

Mentions: Several plant hexokinases were cloned by their ability to complement hexokinase-deficient yeast strains [61-63]. We previously found that PpHxk1 fails to complement a hxk1 hxk2 glk1 triple mutant yeast strain. We noted that PpHxk1 is a type A hexokinase, while all those that had been shown to work in yeast at that time were type B hexokinases [5]. This prompted us to test if a type B hexokinase from Physcomitrella would work in yeast. To this end, we cloned a cDNA encoding PpHxk3 into the yeast shuttle vector pFL61 where the inserts are expressed from the PGK promoter. The plasmid was transformed into the hxk1 hxk2 glk1 yeast strain and tested for ability to support growth on different carbon sources. As shown in Figure 8, we found that PpHxk3 complements the hexokinase-deficient yeast strain for growth on glucose, which shows that PpHxk3 is expressed and active in yeast. We further found that PpHxk3 can support growth on raffinose, which requires fructokinase activity (Figure 8). This shows that PpHxk3 has a dual specificity for glucose and fructose, similar to PpHxk1 [5]. In contrast, PpHxk1 failed to complement the hxk1 hxk2 glk1 triple mutant when expressed from the same vector (Figure 8). To test if this is due to the presence of the chloroplast transit peptide, which might interfere with its function in yeast, we tested a truncated PpHxk1 which lacks residues 1-38. This is the same truncation that causes the PpHxk1-GFP fusion to localize to the cytosol instead of to the chloroplasts [5]. However, the truncated PpHxk1 was still unable to complement the hexokinase-deficient yeast strain (Figure 8). This is in contrast to the type A hexokinases OsHxk4 and LeHxk4 which could complement a hexokinase-deficient yeast strain when their chloroplast transit peptides were deleted [7,12].


Two novel types of hexokinases in the moss Physcomitrella patens.

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

PpHxk3 can complement a hexokinase-deficient yeast strain. The picture shows growth of the hxk1 hxk2 glk1 triple disrupted yeast strain containing the pFL61 vector with different inserts on plates containing 2% galactose, 2% glucose or 3% raffinose as carbon source. Growth on glucose requires glucokinase activity and growth on raffinose fructokinase activity. The inserts from left to right are: PpHXK3 cDNA; PpHXK1 cDNA encoding an N-terminally truncated protein; PpHXK1 cDNA; no insert.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 8: PpHxk3 can complement a hexokinase-deficient yeast strain. The picture shows growth of the hxk1 hxk2 glk1 triple disrupted yeast strain containing the pFL61 vector with different inserts on plates containing 2% galactose, 2% glucose or 3% raffinose as carbon source. Growth on glucose requires glucokinase activity and growth on raffinose fructokinase activity. The inserts from left to right are: PpHXK3 cDNA; PpHXK1 cDNA encoding an N-terminally truncated protein; PpHXK1 cDNA; no insert.
Mentions: Several plant hexokinases were cloned by their ability to complement hexokinase-deficient yeast strains [61-63]. We previously found that PpHxk1 fails to complement a hxk1 hxk2 glk1 triple mutant yeast strain. We noted that PpHxk1 is a type A hexokinase, while all those that had been shown to work in yeast at that time were type B hexokinases [5]. This prompted us to test if a type B hexokinase from Physcomitrella would work in yeast. To this end, we cloned a cDNA encoding PpHxk3 into the yeast shuttle vector pFL61 where the inserts are expressed from the PGK promoter. The plasmid was transformed into the hxk1 hxk2 glk1 yeast strain and tested for ability to support growth on different carbon sources. As shown in Figure 8, we found that PpHxk3 complements the hexokinase-deficient yeast strain for growth on glucose, which shows that PpHxk3 is expressed and active in yeast. We further found that PpHxk3 can support growth on raffinose, which requires fructokinase activity (Figure 8). This shows that PpHxk3 has a dual specificity for glucose and fructose, similar to PpHxk1 [5]. In contrast, PpHxk1 failed to complement the hxk1 hxk2 glk1 triple mutant when expressed from the same vector (Figure 8). To test if this is due to the presence of the chloroplast transit peptide, which might interfere with its function in yeast, we tested a truncated PpHxk1 which lacks residues 1-38. This is the same truncation that causes the PpHxk1-GFP fusion to localize to the cytosol instead of to the chloroplasts [5]. However, the truncated PpHxk1 was still unable to complement the hexokinase-deficient yeast strain (Figure 8). This is in contrast to the type A hexokinases OsHxk4 and LeHxk4 which could complement a hexokinase-deficient yeast strain when their chloroplast transit peptides were deleted [7,12].

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
Related in: MedlinePlus