ABSTRACT
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.
Subject(s)
Peptide Elongation Factor 2/metabolism , Phosphoproteins/metabolism , Ribosomal Proteins/metabolism , Ribosomes/metabolism , Saccharomyces cerevisiae/metabolism , Amino Acid Sequence , Binding Sites/genetics , Chromatography, Affinity , Molecular Sequence Data , Precipitin Tests , Protein Binding , Saccharomyces cerevisiae/genetics , Two-Hybrid System TechniquesABSTRACT
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.
Subject(s)
Amino Acid Motifs/physiology , Carboxylic Acids/metabolism , Cell Compartmentation/physiology , Golgi Apparatus/metabolism , Intracellular Membranes/metabolism , Monosaccharide Transport Proteins/metabolism , Muscle Proteins , 3T3 Cells , Alanine/metabolism , Animals , Cell Membrane/metabolism , Dipeptides/metabolism , Endosomes/metabolism , Enzyme Inhibitors/pharmacology , Genistein/pharmacology , Glucose Transporter Type 4 , Insulin/metabolism , Mice , Peptide Fragments/metabolism , Peptides/metabolism , Phenylalanine/metabolism , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/drug effects , Tyrosine/metabolism , rab GTP-Binding Proteins/metabolism , rab7 GTP-Binding ProteinsABSTRACT
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.
Subject(s)
Poly C/metabolism , Poly U/metabolism , Ribonucleases/isolation & purification , Ribonucleases/metabolism , Saccharomyces cerevisiae/enzymology , Chromatography, Affinity , Chromatography, Ion Exchange , Electrophoresis, Polyacrylamide Gel , Hydrogen-Ion Concentration , Kinetics , Molecular Weight , Polyribonucleotides/metabolism , RNA, Messenger/metabolism , RNA, Ribosomal/metabolism , RNA, Transfer/metabolism , Ribonucleases/chemistry , Substrate Specificity , Transcription, GeneticABSTRACT
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.
