Characterization of interaction sites in the Saccharomyces cerevisiae ribosomal stalk components.

The interactions among the yeast stalk components (P0, P1alpha, P1beta, P2alpha and P2beta) and with EF-2 have been explored using immunoprecipitation, affinity chromatography and the two-hybrid system. No stable association was detected between acidic proteins of the same type. In contrast, P1alpha and P1beta were found to interact with P2beta and P2alpha respectively. An interaction of P0 with P1 proteins, but not with P2 proteins, was also detected. This interaction is strongly increased with the P0 carboxyl end, which is able to form a pentameric complex with the four acidic proteins. The P1/P2 binding site has been located between residues 212 and 262 using different C-terminal P0 fragments. Immunoprecipitation shows the association of EF-2 with protein P0. However, the interaction is stronger with the P1/P2 proteins than with P0 in the two-hybrid assay. This interaction improves using the 100-amino-acid-long C-end of P0 and is even higher with the last 50 amino acids. The data indicate a specific association of P1alpha with P2beta and of P1beta with P2alpha rather than the dimerization of the acidic proteins found in prokaryotes. In addition, they suggest that stalk assembly begins by the interaction of the P1 proteins with P0. Moreover, as functional interactions of the complete P0 were found to increase using protein fragments, the data suggest that some active sites are exposed in the ribosome as a result of conformational changes that take place during stalk assembly and function.

Recycling of the insulin-sensitive glucose transporter GLUT4. Access of surface internalized GLUT4 molecules to the perinuclear storage compartment is mediated by the Phe5-Gln6-Gln7-Ile8 motif.

The insulin-sensitive glucose transporter GLUT4 is translocated to the plasma membrane in response to insulin and recycled back to the intracellular store(s) after removal of the hormone. We have used clonal 3T3-L1 fibroblasts and adipocyte-like cells stably expressing wild-type GLUT4 to characterize (a) the intracellular compartment where the bulk of GLUT4 is intracellularly stored and (b) the mechanisms involved in the recycling of endocytosed GLUT4 to the store compartment. Surface internalized GLUT4 is targeted to a large, flat, fenestrated saccular structure resistant to brefeldin A that localized to the vicinity of the Golgi complex is sealed to endocytosed transferrin (GLUT4 storage compartment). Recycling of endocytosed GLUT4 was studied by comparing the cellular distributions of antibody/biotin tagged GLUT4 and GLUT4(Ser(5)), a mutant with the Phe(5)-Gln(6)-Gln(7)-Ile(8) inactivated by the substitution of Ser for Phe(5). Ablation of the Phe(5)-Gln(6)-Gln(7)-Ile(8) inhibits the recycling of endocytosed GLUT4 to the GLUT4 store compartment and results in its transport to late endosomes/lysosomes where it is rapidly degraded.

Targeting motifs in GLUT4 (review).

The trafficking of the insulin-sensitive glucose transporter, GLUT4, is the paradigm of how cells control the movement of membrane proteins through intricate pathways of transport in response to external stimuli, and how, by doing so, regulate their function. The GLUT4 intracellularly sequestered in resting adipocytes and muscle cells becomes exposed on their surface in response to an increase in insulin levels and muscle contraction, where it facilitates glucose uptake. Ceasing of the stimuli is followed by endocytosis of the GLUT4 molecules exposed on the plasma membrane and their recycling to the original stores, where they are retained. This review discusses current understanding of the organelles that host GLUT4 and the motifs that mediate its trafficking.

Intracellular targeting and retention of the glucose transporter GLUT4 by the perinuclear storage compartment involves distinct carboxyl-tail motifs.

The mechanisms by which the insulin-sensitive glucose transporter, GLUT4, is targeted and retained in a storage compartment near to the Golgi complex are poorly understood. Here we report that removal of the carboxyl-terminal acidic Pro(505)AspGluAsnAsp(509) sequence prevents the storage of GLUT4 in the VAMP-2 positive compartment adjacent to the Golgi complex (GSC), and results in its targeting to GLUT4-positive vesicles and Rab7-positive late endosomes. Storage of the truncated GLUT4 in the GSC is restored by substitution of Phe for the Tyr(502) residue adjacent to Pro(505) or by treatment of cells with the tyrosine kinase inhibitor genistein. Ablation of the Leu(489)Leu(490)-based motif prevents the targeting of GLUT4delta5 to GLUT4-positive-vesicles and late endosomes as well as the retention of GLUT4delta5Phe(502 )by the GSC. These results are consisting with a model of GLUT4 transport in which the targeting of the protein from the TGN to the GSC is mediated by the Leu(489)Leu(490)-based motif and its release from the GSC involves Tyr(502 )and the adjacent carboxyl-terminal Pro(505)AspGluAsnAsp(509) sequence.

Purification and characterization of a novel poly(U), poly(C) ribonuclease from Saccharomyces cerevisiae.

A new ribonuclease from Saccharomyces cerevisiae, specific for poly(U) and poly(C) substrate, was purified near to homogeneity by successive fractionation with DEAE-Sepharose, Heparin-Sepharose and CM-Sepharose chromatography. The purified molecule detected by SDS/polyacrylimide gel electrophoresis has a molecular mass of 29 kDa. The optimum pH for the enzyme activity is 5.5-7 and its isoelectric point is 7.5. The purified enzyme was able to degrade 26S, 18S and 5S rRNAs as well as mRNA obtained from in vitro transcription. No catalytic activity was observed when the RNase was incubated with tRNA and double stranded substrate. Our findings suggest that this novel RNase may play an important role in the processing of RNA in Saccharomyces cerevisiae.

In vitro translation of an intact mRNA coding for a poly(U), poly(C) specific ribonuclease isolated from six-day-old larvae of Ceratitis capitata by a modified extraction procedure.

Intact ribonucleic acid was prepared from six-day-old larvae of Ceratitis capitata, by enriching the guanidinium thiocyanate extraction procedure with a specific mixture for the active ribonuclease inhibition. RNA obtained by this means was then used as a source for the identification of mRNA coding for poly(U), poly(C) specific ribonuclease. The isolated poly(A+) RNA was translated in a cell-free protein synthesizing system. The presence of a poly(U), poly(C) ribonuclease among the newly synthesized products was detected by immunoprecipitation with anti-rabbit polyclonal antibodies against poly(U), poly(C) ribonuclease.