A cDNA Encoding a Membrane Protein from Sugarcane

A sugarcane (Saccharum spp.) leaf cDNA library was screened with a 1.4-kb EcoRV-BsmI fragment of pTF414A (Glc transporter cDNA from Arabidopsis thaliana), which includes most of the coding region of the cDNA (Sauer et al., 1990). Plaque hybridizations were performed at low stringency with the random primed 32P-labeled fragment of pTF414A followed by exposure of the nitrocellulose membranes to film for 48 h at -7OOC. The cDNA of one of the positive clones was subcloned and sequencing revealed a cDNA of 1279 nucleotides. This cDNA (SMPl) contained an open reading frame encoding a polypeptide of 325 amino acids with a predicted molecular mass of 35.8 kD (Table I). The nucleotide sequence around the first Met of this open reading frame has 67% homology to the consensus translation initiation site of plants (Joshi, 1987). Hydropathy analysis indicated that the SMPl protein is a putative membrane protein. The grand average hydropathy value of the SMPl protein was 0.38, significantly greater than the mean for soluble proteins (-0.4) (Kyte and Doolittle, 1982). This value is similar to values for membrane transport proteins for Glc (0.48) (Sauer et al., 1990) and SUC (0.57) (Riesmeier et al., 1992). In addition, SMPl protein has a calculated isoelectric point (8.62) that is similar to that of the membrane transport proteins for Glc (9.49) and SUC (8.81). Furthermore, a hydropathy plot revealed that the deduced protein contains six putative membrane-spanning regions of 19 to 26 amino acids, with an average hydropathy index of >1.6 (Kyte and Doolittle, 1982). The protein encoded by SMPl does not share any significant overall homology with proteins in the GenBank, EMBL, and Swiss Protein data bases (Pearson and Lipman, 1988). The SMPl sequence is 38% homologous at the nucleotide level to the fragment of the Glc transporter (pTF414A) used for screening the library, and shares 21% identity at the amino acid level with the Glc transporter.