Tata Institute of Fundamental Research

Diversity of GTP-binding proteins (GPs) in corn and sorghum
--Panigrahi, KC; Johri, MM

In plants, GTP-binding proteins have been suggested to mediate the signal transduction process in response to external stimuli. Towards this ultimate aim, we have examined the dark-grown coleoptiles of corn and sorghum for the presence of various GPs using immunological and biochemical methods. Based on the binding of 32[P]-€-GTP to the polypeptides on the western blots, cholera toxin (CT) catalysed ADP-ribosylation in the presence of GTP, and cross reactivity with anti-G-€-common antibodies, three classes of GPs were observed (Table 1). These are: i) low Mr GPs of 18-29 kDa, ii) high Mr GPs of 32-45 KDa which seem to be the €-subunits of heterotrimeric GPs and iii) another class of high Mr GPs of 56-74 KDa. The G-€-common polyclonal antibodies were produced using the GTP-binding domain peptide conjugated to KLH.

These studies show that the antibodies cross react with all classes of GPs and thus the immunodetection method is far more versatile as compared to the other two methods. Most of the low Mr GPs were detectable when GTP-binding method was used, while only a subset of high Mr GPs were detectable on the basis of CT-enhanced ADP ribosylation. Most of the GPs seem to be conserved and only a few are species-specific. Whereas corn shows a single high Mr GP (56-60 kDa), sorghum contained three different ones of which two were also ADP-ribosylated. The ADP-ribosylation of the €-subunit of heterotrimeric GPs is well known and such heterotrimeric GPs are localized in the plasmamembrane. In the present studies these GPs were found to be present not only in the microsomal pellet but also with the cytosolic and organellar fractions. At least four different ones were observed in corn and of these two were ADP ribosylated in the presence of CT. The sizes of corn GPs found in present studies are comparable to that reported earlier (see Ma, Plant Mol. Biol. 26:1611, 1994). The sorghum on the other hand has only three types of €-subunits.

We are trying to determine if the low Mr GPs belong to the Ras, Rho or Ypt/Rab class, which are known to play a role in secretion, intracellular transport or the restructuring of cytoskeleton during development and differentiation. In addition to the €-subunit of heterotrimeric GPs, the present studies suggest that some of the GPs of still higher Mr (56-60 and 68 kDa) can also be ADP-ribosylated in vitro. High Mr GPs have earlier been reported in mammalian systems; for instance, a 67 KDa GP has been reported to be coupled to insulin receptor and plays a role in the signal transduction process. A similar function for the higher Mr GP in plants cannot be ruled out.

Table 1. Different types of GTP-binding proteins from corn and sorghum.

Size (KDa)Detection methodsSub-cellular localizationPlant species
68Ab, AROrganellesS
56-60Ab, AROrganellesC, S
44-45Ab, ARMicrosomes and cytosolC, S
41-43Ab, AROrganelles, microsomes and cytosolC, S
35-38.5AbMicrosomes and cytosolC, S
32AbMicrosomes and cytosolC
29,27,25*,18Ab, 32P-GTPMicrosomesC
29,26,18Ab, 32P-GTPChloroplastsC, S

Ab - Polypeptides recognised by G-€ common antibodies; AR - Polypeptides ADP-ribosylated; 32P-GTP - Polypeptides binding labelled GTP. C - corn and S - sorghum.
*Small Mr GPs not detected by the GTP-binding method.

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