1. Identification of ER structuring proteins

The identification of ER structuring proteins is being achieved in our lab using a cell-based RNA interference (RNAi) approach where individual candidate proteins are targeted by RNAi and the consequence on ER structure and organization explored using light and
 electron microscopic analyses. Currently, our lab is focused on two proteins whose depletion alters ER morphology in dramatically distinct ways. The first is Yip1A, a five-pass transmembrane protein with no known enzymatic function but whose depletion leads to loss of ER dispersal and stacking of ER membranes into concentric membrane whorls. The second protein of interest is atlastin, a membrane-anchored large GTPase whose depletion leads to a severe reduction in ER network interconnections. Both proteins are highly conserved, and in the case of atlastin, its mutation in neurons is linked to an autosomal dominant form of hereditary spastic paraplegia (HSP), a human motor neurological disorder.

 

2. Structure function analysis of ER shaping proteins

Functional replacement by an RNAi-immune trans-gene encoding the protein of interest lays the foundation for structure function analysis. In the case of the atlastin GTPase, mutations informed by prior structure determinations are made and the effects of the mutations on the protein’s function discerned in the native environment of cells. Mutations that impair function in cells are analyzed in parallel using a variety of pure protein biochemical and biophysical assays in the context of synthetic membranes. Merging the outcomes of ‘in vivo’ and ‘in vitro’ assays provides insights into functionally important catalytic residues and conformational changes used by the structuring proteins to shape ER membranes.

 

3. Understanding the role of ER structuring proteins in disease

The shape, organization and dynamics of the ER network are likely to be intimately intertwined with the function of the organelle. Consequently, aberrant ER morphology due to loss of a required structuring protein is predicted to lead to organelle dysfunction. As a case in point, mutations in the neuronal atlastin1 GTPase lead to the degeneration of long axons in the corticospinal tract thereby causing the motor neurological disorder HSP. However, the cell biological basis for axon degeneration is not clearly understood. Work in our lab also aims to better understand the link between ER structure and diseases such as HSP.