Pumpkin eIF5A isoforms interact with components of the translational machinery in the cucurbit sieve tube system

Abstract

SUMMARYIn yeast, eIF5A, in combination with eEF2, functions at the translation step, during the protein elongation cycle. This result is of significance with respect to functioning of the enucleate sieve tube system, as eIF5A was recently detected in Cucurbita maxima (pumpkin) phloem sap. In the present study, we further characterized four CmeIF5A isoforms, encoding three proteins, all of which were present in the phloem sap. Although hypusination of CmeIF5A was not necessary for entry into the sieve elements, this unique post-translational modification was necessary for RNA binding. The two enzymes required for hypusination were detected in pumpkin phloem sap, where presumably this modification takes place. A combination of gel-filtration chromatography and protein overlay assays demonstrated that, as in yeast, CmeIF5A interacts with phloem proteins, like eEF2, known to be involved in protein synthesis. These findings are discussed in terms of a potential role for eIF5A in regulating protein synthesis within the enucleate sieve tube system of the angiosperms.

Mechanism of Cytoplasmic mRNA Translation

“…A number of studies of plant eEF5 have indicated a role in stress responses (abiotic, pathogen, iron deficiency), growth and development (Wang et al, 2003;Chou et al, 2004;Hopkins et al, 2008;Ma et al, 2010;Lan and Schmidt, 2011;. eEF5 was found to be associated with eEF2 in pumpkin phloem .…”

Overexpression of Arabidopsis Plasmodesmata Germin-Like Proteins Disrupts Root Growth and Development

“…Purification of recombinant proteins from ZYMV viral vector-infected pumpkin leaves was performed as described previously (Ma et al, 2010;Li et al, 2011). Briefly, ZYMV-based constructs were coated onto gold particles (1 µm) and bombarded onto pumpkin cotyledons using a Helios gene gun system (Bio-Rad).…”

“…Genotypes of these mutants were confirmed by PCR analysis using the appropriate primer sets (see Supplemental Table 2 online). Pumpkin (Cucurbita maxima cv Big Max) and Nicotiana benthamiana plants were grown as described previously (Taoka et al, 2007;Ma et al, 2010) for use in recombinant protein expression and (A) Control (Vec), pdglp1 pdglp2 double mutant, P PDGLP1 :PG1-GFP, P PDGLP2 :PG2-GFP, P 35S :PDGLP1-Myc, and P 35S :PDGLP2-Myc plants were grown vertically on Murashige and Skoog plates for 8 d and then plates were rotated 90°to test the capacity of these lines to undergo differential growth in response to the altered gravitropic signal. Note that the primary roots of all tagged plant lines displayed a normal gravitropic response.…”

“…For pull-down assays, GFP, Nt-NCAPP1DN 1-22 , PDGLP1, and PDGLP1DSP were subcloned into a Zucchini yellow mosaic virus (ZYMV)-based viral vector as described previously (Ma et al, 2010;Li et al, 2011). GST and Cm-PP16-GST were amplified from pGWB24/CmPP16 (Li et al, 2011) and inserted into the modified ZYMV viral vector without a c-MycX4-His 6 tag (Lin et al, 2002).…”

Cloning and expression analysis ofeIF-5Agene inApocynum venetum

“…[8] The previous reports about eIF-5A are largely focused on human [9] and yeast cells, [10] but the precise cellular function of eIF-5A remains largely unknown, especially in plants. Although the full-length cDNA encoding eIF-5A has been isolated from several plant species such as Arabidopsis thaliana,[11À13] pumpkin, [14] maize, [15] tomato, [16] Tamarix androssowii [17] and so on, the function and mechanism of this gene in higher plants are not clear, especially its biological function under abiotic stress.…”