Two newly identified membrane-associated and plastidic tomato HXKs: characteristics, predicted structure and intracellular localization

Abstract

Two new tomato hexokinase genes, LeHXK3 and LeHXK4, were cloned and characterized, placing tomato as the first plant with four characterized HXK genes. Based on their sequence, LeHXK3 is the third membrane-associated (type-B) and LeHXK4 is the first plastidic (type-A) HXK identified in tomato. Expression of HXK-GFP fusion proteins in protoplasts indicated that the LeHxk3 enzyme is associated with the mitochondria while LeHxk4 is localized in plastids. Furthermore, LeHxk4::GFP fusion protein is found within stromules, suggesting transport of LeHxk4 between plastids. Structure prediction of the various plant HXK enzymes suggests that unlike the plastidic HXKs, the predicted membrane-associated HXKs are positively charged near their putative N-terminal membrane anchor domain, which might enhance their association with the negatively charged membranes. LeHxk3 and LeHxk4 were analyzed following expression in yeast. Both enzymes have higher affinity for glucose relative to fructose and are inhibited by ADP. Yet, unlike the other HXKs, the stromal HXK has higher Vmax with glucose than with fructose. Expression analysis of the four HXK genes in tomato tissues demonstrated that LeHXK1 and LeHXK4 are the dominant HXKs in all tissues examined. Notably, the plastidic LeHXK4 is expressed in all tissues including starchless, non-photosynthetic sink tissues, such as pink and red fruits, implying phosphorylation of imported hexoses in plastids. It has been suggested that trehalose 6-phosphate (T6P) might inhibit HXK activity. However, none of the yeast-expressed tomato HXK genes was sensitive either to T6P or to trehalose, suggesting that unlike fungi HXKs, plant HXKs are not regulated by T6P.

Expression and evolutionary features of the hexokinase gene family in Arabidopsis

“…2). As noted with other plastid HXKs (Cho et al 2006a;Kandel-Kfir et al 2006), the presence of the transit peptide in AtHXK3 might have led to this protein not being identified in a previous complementation study of yeast cells deficient in glucose phosphorylation activity, using Arabidopsis cDNAs (Jang et al 1997). The organ distribution of AtHXK3 is similar to that observed for tobacco plastid NtHXK2 from promoter-GUS studies (Giese et al 2005).…”

Trehalose 6-Phosphate Is Required for the Onset of Leaf Senescence Associated with High Carbon Availability

“…Whether T6P is involved in hexokinase signaling is currently unclear. Whereas yeast hexokinase is inhibited by T6P, no such effect has been found for plants (Wiese et al, 1999;Eastmond et al, 2002;Kandel-Kfir et al, 2006), and the impact of T6P on gene expression in seedlings was considered to be largely independent of hexokinase signaling (Zhang et al, 2009). Nevertheless, effects of hexokinase-1 on TPS1 expression in Arabidopsis seedlings were demonstrated by Avonce et al (2004), suggesting that there may be signaling interactions.…”

Sugar Sensing and Signaling

“…Proteomic and GFP fusion analyses indicate that several plant HXKs are located on the outer mitochondrial membrane, where a completely functional glycolytic metabolon can be found (Giege et al, 2003;Kim et al, 2006;Balasubramanian et al, 2007;Damari-Weissler et al, 2007;Granot, 2007;Karve et al, 2008). In addition, plant HXKs are found in plastids (Wiese et al, 1999;Olsson et al, 2003;Giese et al, 2005;Cho et al, 2006a;Kandel-Kfir et al, 2006;Granot, 2007). Unique in moss, the chloroplast stromal HXK accounts for 80% of the total hexose kinase activity and is responsible for glucose-mediated growth and formation of caulonemal filaments (Olsson et al, 2003).…”