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Investigation into the role of the SEC65 gene product in protein translocation across the yeast ER membrane
Başlık:
Investigation into the role of the SEC65 gene product in protein translocation across the yeast ER membrane
Yazar:
Hewitt, Eric W., author.
ISBN:
9780438043930
Yazar Ek Girişi:
Fiziksel Tanımlama:
1 electronic resource (241 pages)
Genel Not:
Source: Dissertation Abstracts International, Volume: 76-08C.
Özet:
Mammalian signal recognition particle (SRP) is an 11S ribonucleoprotein complex, which targets secretory proteins to the ER membrane. Biochemical reconstitution of protein translocation across the yeast ER membrane did not demonstrate an SRP like activity. However, a number of genes have been isolated which encode putative components of the yeast SRP. The SEC65 gene was cloned by complementation of the sec65-1 temperature sensitive mutation. Analysis of the sequence indicated that it shared significant sequence identity with the 19 kDa subunit of mammalian SRP (SRP19). In addition an extragenic suppresser had been isolated, which at multicopy could suppress the sec65-1 mutation. This corresponded to the previously identified SRP54sc, the yeast homologue of mammalian SRP54. Analysis of these proteins by sucrose gradient sedimentation indicated that they cofractionated with an RNA component (SCR1) in a 17.55 SRP complex. Although larger than mammalian SRP, gel filtration of the yeast particle suggested an extended structure consistent with that observed with its mammalian counterpart. Two observations suggest that SRP54scp and SEC65p interact in the assembly of the yeast SRP complex, this being consistent with the interaction between their respective homologues in the mammalian particle. Firstly, when the sec65-1 mutant was shifted to the restrictive temperature the steady state level of SEC65p was dramatically reduced however, the overexpression of SRP54sc partially stabilised SEC65p. Additionally, when analysed by sucrose gradient sedimentation neither the mutant SEC65p or SRP54scp fractionated in the SRP complex. In order to further characterise the assembly of SEC65p into the yeast SRP, a deletion analysis of the SEC65p sequence was conducted. Sequence alignment between SEC65p and SRP19, suggests the presence of an additional N-terminal domain in SEC65p. However, this region was not required for SEC65p function in vivo, as both this deletion assembled normally with SRP54scp into the particle. This suggests that, the assembly of SEC65p and SRP19 into the SRP complex is conserved. Indeed, C-terminal deletions indicated that only the region which shares significant sequence homology with SRP19 was required for SEC65p's function in vivo, although perhaps not for stable association with the particle. In order to conduct a functional and also a More detailed structural characterisation of the yeast particle, a purification strategy was developed. To this end a histidine tagged SEC65p was generated, and expressed in yeast facilitating the purification of the SRP complex by nickel affinity chromatography. A substantial purification of an intact SRP complex was achieved. Such purified material would be ideal for any further functional characterisation of the yeast SRP complex, within a reconstituted translocation assay. Indeed, analysis of a homologous yeast translocation assay, indicated that translocation of invertase was inefficient, perhaps because SRP activity was limiting. Therefore, purified SRP fractions might be able to stimulate translocation, thereby facilitating the development of an SRP dependent assay.
Notlar:
School code: 1543
Tüzel Kişi Ek Girişi:
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Yer Numarası | Demirbaş Numarası | Shelf Location | Lokasyon / Statüsü / İade Tarihi |
---|---|---|---|
XX(684330.1) | 684330-1001 | Proquest E-Tez Koleksiyonu | Arıyor... |
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