Spinach hexokinase I is located in the outer envelope membrane of plastids

Abstract

The subcellular localization of hexokinase activities in plant cells has been a matter of debate for a long time. We have isolated a hexokinase cDNA fragment from glucose-fed spinach leaves using a differential display reverse transcription-PCR approach. The corresponding cDNA was expressed in Escherichia coli and an antiserum, raised against the recombinant protein, was used in subcellular localization studies. The spinach hexokinase could be localized primarily to the outer envelope membrane of chloroplasts where it is inserted via its N-terminal membrane anchor. We suggest that the chloroplast envelope hexokinase is involved in the energization of glucose export from plastids rather than in the sugar-sensing pathway of the plant cell.z 1999 Federation of European Biochemical Societies.

EST-analysis of the thermo-acidophilic red microalga Galdieriasulphuraria reveals potential for lipid A biosynthesis and unveils the pathway of carbon export from rhodoplasts

“…In support of this hypothesis, we did not find any ESTs with similarity to the plastidic maltose transporter MEX1 (Niittyla et al, 2004), and none of the 13 ESTs encoding putative monosaccharide transporters was closely related to the plastidic glucose translocator pGlcT (Weber et al, 2000). Furthermore, the export of glucose from plastids is likely to require the activity of hexokinase (Wiese et al, 1999;Weber et al, 2000), and antisense repression of hexokinase in potato plants caused a starch excess phenotype (Veramendi et al, 1999). However, as outlined above, we did not find evidence for a eukaryote-type hexokinase in Galdieria, further corroborating the hypothesis that a green plant type night-path for carbon export is absent in Galdieria, and possibly in red algae in general.…”

“…Although the primary structure of red algal glucokinase is not related to hexokinases from green plants, the membrane anchor that is found in several plant hexokinases but not in other eukaryotic hexokinases (Wiese et al, 1999) is also present in the C. merolae enzyme. In contrast to the green plant enzyme that carries the membrane anchor at its N-terminus, the red algal enzyme, however, has a C-terminal membrane anchor.…”

Partial purification of tightly bound mitochondrial hexokinase from maize (Zea mays L.) root membranes

“…Different from studies on cytosolic enzymes, phenyl-Sepharose step was 50 times more efficient in purifying maize NC-HK 1 with similar yields ( Table 1), indicating that this is a suitable step for the purification of NC-HKs. Several studies have been performed with non-cytosolic HKs from different plants (1)(2)(3)(5)(6)(7)(8)(9)(10)(11)(12)(13)28,37). However, since these studies focused on the sub-cellular localization or the determination of the kinetic properties of these enzymes, the current study presents for the first time a procedure for the purification of particulate HKs from plants.…”

“…In fact, the first 24 N-terminal residues of the spinach chloroplast SoHK 1 protein deduced from the cDNA gene are extremely hydrophobic (13), conferring to this plant HK a longer and more hydrophobic N-terminal region than the one found in rat brain HK. Analysis by a hydropathy plot (39) of spinach and rice HK cDNA genes (SoHK 1 and OsHK 1, respectively) and a putative maize NC-HK (ZmHK 1) cDNA gene revealed the presence of a similar hydrophobic N-terminal sequence in these enzymes (Figure 4).…”

“…This observation is supported by the fact that truncated spinach SoHK 1 without 24 N-terminal residues does not associate with the outer chloroplast envelope membrane (13). Wiese et al (13) suggested that this hydrophobic N-terminal is immersed directly into the membrane independently of the presence of any receptor protein. Similar to spinach SoHK 1, it was found that the rice OsHK 1 gene product is associated with the mitochondrial membrane and also presents the first 24 hydrophobic N-terminal residues (28).…”