Messenger RNA-binding properties of nonpolysomal ribonucleoproteins from heat-stressed tomato cells

Most cells experiencing heat stress reprogram their translational machinery to favor the synthesis of heat-stress proteins. Translation of other transcripts is almost completely repressed, but most untranslated messengers are not degraded. In contrast to yeast, Drosophila melanogaster, and HeLa cells, plant cells store repressed messengers in cytoplasmic nonpolysomal ribonucleoproteins (RNPs). To follow the fate of untranslated transcripts, we studied protein composition, mRNA content, and RNA-binding properties of nonpolysomal RNPs from heat-stressed tomato (Lycopersicon peruvianum) cells. Contrary to the selective interaction in vivo, RNPs isolated from tomato cells bound both stress-induced and repressed messengers, suggesting that the selection mechanism resides elsewhere. This binding was independent of a cap or a poly(A) tail. The possible role of proteasomes and heat-stress granules (HSGs) in mRNA storage is a topic of debate. We found in vitro messenger-RNA-binding activity in messenger RNP fractions free of C2-subunit-containing proteasomes and HSGs. In addition, mRNAs introduced into tobacco (Nicotiana plumbaginifolia) protoplasts were found in the cytoplasm but were not associated with HSGs.

The hexose transporters at the plasma membrane and the tonoplast of transformed plant cells: kinetic characterization of two distinct carriers.

The plasma membrane hexose transporter and the tonoplast hexose transporter from heterotrophically grown transformed Nicotiana tabacum cells have been studied in vitro using membrane vesicles for trans-zero transport studies. In highly purified phase-partitioned outside-out plasma membrane vesicles (PMV) the hexose transporter showed an apparent Km value of 230 microM (substrate: 3-O-methyl-D-glucose (3-OMG); pHi 7.2/pHo 7.2), which was reduced to 120 microM when a pH gradient was imposed (pHo 5.7/pHi 7.2). However, the Vmax value was not affected indicating that no stable pH gradient was formed. Uptake experiments with 14C-labelled acetate supported this interpretation. Transport was insensitive to N-ethylmaleimide (NEM; up to 1 mM concentration) and p-chloromercuribenzene sulfonate (PCMBS; up to 500 microM), whereas the tonoplast hexose transporter (in mixed inside / out and outside / out vesicles) was inhibited by NEM in a substrate-protectable manner, and PCMBS was also inhibitory. Kinetically two components with apparent Km values of 6 and 20 mM could be distinguished for the tonoplast hexose transporter. Substrate specificities of both transporters were similar except for D-galactose and D-fructose. The results indicate structural differences between the tonoplast and plasma membrane hexose transporters in plants.