RESUMO
A 17 kDa polypeptide found in association with actin in cellular extracts of Dictyostelium discoideum was identified as a proteolytic fragment of eEF1beta. Antibody elicited against the 17 kDa protein reacted with a single 29 kDa polypeptide in Dictyostelium, indicating that the 17 kDa peptide arises from degradation of a larger precursor. The cDNA isolated from a Dictyostelium library using this antibody as a probe encodes Dictyostelium elongation factor 1beta. Amino acid degradation of the 17 kDa protein fragment confirmed the identity of the protein as eEF1beta. Direct interaction of eEF1beta with actin in vitro was further demonstrated in mixtures of actin with the 17 kDa protein fragment of Dictyostelium eEF1beta, recombinant preparations of Dictyostelium eEF1beta expressed in Escherichia coli, and the intact eEF1betagamma complex purified from wheat germ. Localization of eEF1beta in Dictyostelium by immunofluorescence microscopy reveals both diffuse cytoplasmic staining, and some concentration in the cortical and hyaline cytoplasm. The results support the existence of physical and functional interactions of the translation apparatus with the cytoskeleton, and suggest that eEF1beta may function in a dual role both to promote the elongation phase of protein synthesis, and to interact with cytoplasmic actin.
Assuntos
Dictyostelium/química , Proteínas dos Microfilamentos/química , Fator 1 de Elongação de Peptídeos/química , Actinas/química , Actinas/isolamento & purificação , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Sequência de Bases , Clonagem Molecular , Citoesqueleto/química , Dictyostelium/genética , Biblioteca Gênica , Proteínas dos Microfilamentos/biossíntese , Proteínas dos Microfilamentos/genética , Microscopia de Fluorescência , Dados de Sequência Molecular , Fator 1 de Elongação de Peptídeos/genética , Fator 1 de Elongação de Peptídeos/imunologia , Mapeamento por Restrição , Alinhamento de SequênciaRESUMO
In protein transport between organelles, interactions of v- and t-SNARE proteins are required for fusion of protein-containing vesicles with appropriate target compartments. Mammalian SNARE proteins have been observed to interact with NSF and SNAP, and yeast SNAREs with yeast homologues of NSF and SNAP proteins. This observation led to the hypothesis that, despite low sequence homology, SNARE proteins are structurally similar among eukaryotes. SNARE proteins can be classified into two groups depending on whether they interact with SNARE binding partners via conserved glutamine (Q-SNAREs) or arginine (R-SNAREs). Much of the published structural data available is for SNAREs involved in exocytosis (either in yeast or synaptic vesicles). This paper describes circular dichroism, Fourier transform infrared spectroscopy, and dynamic light scattering data for a set of yeast v- and t-SNARE proteins, Vti1p and Pep12p, that are Q-SNAREs involved in intracellular trafficking. Our results suggest that the secondary structure of Vti1p is highly alpha-helical and that Vti1p forms multimers under a variety of solution conditions. In these respects, Vti1p appears to be distinct from R-SNARE proteins characterized previously. The alpha-helicity of Vti1p is similar to that of Q-SNARE proteins characterized previously. Pep12p, a Q-SNARE, is highly alpha-helical. It is distinct from other Q-SNAREs in that it forms dimers under many of the solution conditions tested in our experiments. The results presented in this paper are among the first to suggest heterogeneity in the functioning of SNARE complexes.
Assuntos
Proteínas de Transporte/química , Proteínas de Membrana/química , Proteínas de Saccharomyces cerevisiae , Proteínas de Transporte Vesicular , Sequência de Aminoácidos , Animais , Clonagem Molecular , Proteínas Fúngicas/química , Luz , Mamíferos , Dados de Sequência Molecular , Conformação Proteica , Estrutura Secundária de Proteína , Proteínas Qa-SNARE , Proteínas Qb-SNARE , Proteínas Recombinantes , Saccharomyces cerevisiae/metabolismo , Espalhamento de RadiaçãoRESUMO
The ciliated protozoan, Tetrahymena thermophila, offers an attractive medium for the expression of heterologous proteins and could prove particularly useful for the display of foreign proteins on the cell surface. Although progress has been made in transformation of Tetrahymena with heterologous DNA, methods that permit reliable expression of foreign genes have been lacking. Using a mutant strain of T. thermophila carrying a negatively selectable allele of a beta-tubulin gene, we have been able to direct foreign genes to this locus by homologous recombination. Transformed cell lines producing foreign proteins were readily identified and, in at least one case, targeting of proteins to the plasma membrane was accomplished.
