Lee, Hannah, Ph.D. (2012) Factors regulating endoplasmic reticulum morphology and quality control. PhD thesis, University of Warwick.

Abstract

The ER is a network of membrane sheets and tubules connected via three-way junctions. Reticulons are integral membrane proteins responsible for shaping the tubular ER. There are four reticulons isoforms in animals, two in yeast and 21 isoforms in Arabidopsis. This study developed an expression profile for isoforms AtRTNLB1-18 by expressing these reticulons driven by their native promoters. The reticulons analysed localised to the ER and coexist in many tissues. Given this result it is possible that the large size of the Arabidopsis gene family may have evolved to afford functional redundancy. It is known that a knock-out of seed specific RTNLB13 does not disrupt the seed ER morphology. Is this due to functional redundancy? In this study knock-outs were made of two other reticulons found non-exclusively in the seed, RTNLB1 and RTNLB2. In accordance with the redundancy theory, there was no apparent disruption in the mutants ER morphology. Knock-out/knock-down mutants of RTNLB1, RTNLB2 and RTNLB13 were also made and verified. The results of this were beyond the time frame of this study. In mammals and yeast reticulons are known to interact with other tubule forming proteins (DP1/Yop1p) and human atlastin. The Arabidopsis homologue of Yop1p is HVA22. In this study the seed-specific isoform HVA22b labels the tobacco ER (and the nuclear envelope), but it does not alter ER morphology or cause constrictions of the tubules (as seen with RTNLB13), suggesting that it is not a structural component. The closest plant homologue of atlastin is RHD3. This study shows that RL2, an RHD3 isoform that is highly expressed in the seed, locates to the ER without perturbing the ER morphology or Golgi body mobility. Over-expression of RL2 bearing mutations within its GTPase domain, however, induces cable-like ER, suggesting that a functional GTPase domain is required for the formation of three-way junctions. Co-expression of RTNLB13 with RL2 resulted in a striking modification of the ER network. This alteration was independent of an active RL2 GTPase domain but required a functional reticulon. RL2 and its GTPase mutants co-immunoprecipitate with RTNLB13. These results indicate that RL2 and RTNLB13 interact and operate synergistically in modulating ER morphology.

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