Cysteine residues of the mammalian GET receptor: Essential for tail-anchored protein insertion?

by Moritz Schaefer
Doctoral thesis
Date of Examination:2017-05-30
Date of issue:2017-05-17
Advisor:Prof. Dr. Blanche Schwappach
Referee:Prof. Dr. Michael Thumm
Referee:Prof. Dr. Martin Oppermann
Persistent Address: http://dx.doi.org/10.53846/goediss-6307

 

 

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Abstract

English

Tail-anchored proteins integrate into lipid bilayers via a single transmembrane segment at their extreme C-terminus. The membrane insertion step is mediated by a heterodimeric receptor complex formed by WRB and CAML in higher eukaryotes and Get1 and Get2 in yeast. The transmembrane domains of the receptor complex form an integration site at the endoplasmic reticulum membrane and interact transiently with the transmembrane domain of an integrating TA protein prior to its membrane embedding. Cysteine residues are absent in yeast Get1 and Get2 whereas the mammalian homologues WRB and CAML contain three and five cysteines respectively. I aimed to investigate the role of the cysteine residues of WRB and CAML and test whether they are essential for TA protein targeting. Mutants of WRB and CAML in which the cysteine residues were mutated to serine were generated and I used a yeast-based complementation assay in which cells depleted of Get1 and Get2 were transformed with plasmids encoding either wild type or mutant WRB and CAML. Mutants were still able to create a receptor complex as assayed by yeast two-hybrid and did not impair the targeting of a substrate TA protein. Moreover, mutants rescued growth phenotypes of GET mutants confirming the formation of a functionally active receptor. In line with the absence of cysteines in the yeast GET receptor, cysteines in WRB and CAML seem to be not essential for TA protein targeting and may be required for potential still unknown regulation of TA protein targeting such as receptor stoichiometry, substrate quality control and chaperone function.
Keywords: GET; Guided entry of tail-anchored proteins; WRB; CAML; GET receptor; Yeast two-hybrid; redox regulation; oxidative stress
 
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