Biochemical and genetic analysis of a fission yeast ribonucleoprotein homologous to signal recognition particle
Brennwald, Patrick Joseph
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Permalink
https://hdl.handle.net/2142/19215
Description
Title
Biochemical and genetic analysis of a fission yeast ribonucleoprotein homologous to signal recognition particle
Author(s)
Brennwald, Patrick Joseph
Issue Date
1990
Doctoral Committee Chair(s)
Wise, Jo Ann
Department of Study
Biochemistry
Discipline
Biochemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Molecular
Biology, Microbiology
Chemistry, Biochemistry
Language
eng
Abstract
Protein translocation across the endoplasmic reticulum of higher cells is dependent on a ribonucleoprotein complex known as signal recognition particle (SRP). SRP is composed of six polypeptides and one molecule of 7SL RNA. A homolog of mammalian SRP RNA was found in the fission yeast, Schizosaccharomyces pombe. The gene encoding the S. pombe RNA was cloned by screening a genomic library. Sequence analysis shows that the two RNAs are structurally similar. Disruption of the chromosomal copy of the single copy 7SL gene is lethal to the haploid yeast, demonstrating that the RNA is essential for cell viability. Since secretion is an essential function of the cell, this observation is consistent with its proposed role in this process.
The S. pombe RNA is part of a ribonucleoprotein particle which shows striking similarity to SRP. The fission yeast complex is, like mammalian SRP, 11S in size and behaves similarly to this particle on both ion exchange and hydrophobic columns. More importantly the S. pombe particle, like canine SRP, is found in the cytoplasm associated with membranes and polysomes in a salt-dependent manner. These properties were used to partially purify the fission yeast particle; several polypeptides were shown to copurify with the RNA moiety. Polyclonal antibodies have been raised against the fission yeast homolog of the 54kD SRP protein which was cloned and sequenced by another laboratory. The predicted molecular weight of the fission yeast homolog (57kD) precisely matches the size of one of the polypeptides observed in the purification. Immunoblot experiments show that this protein is also salt-extractable from membranes and that it cofractionates with fission yeast 7SL RNA on a sucrose gradient. Native immunoprecipitations with antibodies directed against this protein component also precipitate 7SL RNA; they therefore are clearly part of the same complex. The conservation of two components of the mammalian particle is strong evidence that the function of this particle has also been conserved in fission yeast.
In order to test the function of the particle directly a homologous fission yeast in vitro translation/translocation system has been developed. Two model secretory proteins were used: the budding yeast mating pheromone prepro$\alpha$factor and fission yeast pre-acid phosphatase. As in the budding yeast system, prepro$\alpha$factor was capable of post-translationally translocating in the homologous fission yeast system. However fission yeast pre-acid phosphatase was found to be imported only when fission yeast microsomes were present during the translation. This work sets the stage to directly test whether fission yeast SRP functions in either mode of translocation.
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