[Thesis]. Manchester, UK: The University of Manchester; 2012.
During or following mRNA translation by cytosolic ribosomes the nascent protein being
synthesised can be targeted to the endoplasmic reticulum (ER). This targeting of nascent
proteins leads to either their transport across the ER or in the case of transmembrane
proteins their integration into the ER membrane. This process is carried out by the
Sec61 complex which is conserved across organisms including bacteria, yeast and mammals.
The ER is a site of protein folding, modification, forward transport and quality control.
Misfolded proteins can be directed to the ER associated degradation pathway which
mediates retrotranslocation of proteins out of the ER and degradation by the proteasome.
A lot is already known about ER associated processes, however, the function of many
factors associated with the ER are still poorly understood. Two examples of such factors
in yeast include Ysy6p and the ER membrane complex (EMC). The mammalian homolog of
Ysy6p, RAMP4 has been shown to be recruited to ribosomes during the integration of
transmembrane proteins into the ER and implicated in processes such as protein degradation.
The function of the EMC is unknown but has been suggested to function in either protein
folding, forward trafficking from the ER or ERAD.Here it has been shown that deletion
of EMC1, EMC2, EMC3, EMC6 but not EMC4 or EMC5 causes defects in growth at 39.5Â°C
or on media containing SDS. Defective growth on media containing SDS is suggestive
of defects in cell wall or membrane biogenesis and both SDS and elevated temperature
are known to cause the induction of the unfolded protein response. This suggests that
certain members of the EMC complex are involved in stress responses or secretory processes.
Functional investigation of ER associated pathways showed that a âˆ†emc5 strain has
a defect in clearance of the ERAD-M substrate Hmg2-6myc however âˆ†emc1 does not. The
ERAD and competitive growth phenotypes are therefore unlinked and suggests that the
EMC complex has a function in multiple pathways which are independently affected by
deletion of individual members of the complex.To test whether Ysy6p was a functional
yeast homolog of RAMP4, processes previously observed to be involved with the function
of RAMP4 were investigated. Strikingly, it has been demonstrated that Ysy6p, similarly
to RAMP4, is a protein which tightly associates with ribosomes. In addition, RAMP4
has been previously observed to affect protein degradation whereas here it has been
shown that deletion of YSY6 causes an alteration in the degradation profile of Hmg2-6myc.
The findings are therefore consistent with the notion that Ysy6p and RAMP4 are functional
homologs.In order to further characterise the molecular basis for ribosome association
N terminal truncations of opsin tagged Ysy6p were made. It was demonstrated that partial
deletion of a conserved cytosolic domain predicted to form a helix-turn-helix resulted
in a near loss of cosedimentation of Ysy6p with ribosomes. Furthermore, it was shown
that Ysy6p crosslinks with MBS to three factors of 22 kDa, 18 kDa and 10 kDa the identities
of which remain undetermined. The data therefore suggest that Ysy6p is involved in
processes associated with co-translational integration of proteins and ERAD. In addition,
the data also suggest that the EMC complex is involved in ERAD and stress responses.