Project No. 2331

PRIORITY PROJECT

Primary Supervisor

Dr Jan Janouskovec – University of Southampton

Co-Supervisor(s)

Assoc Prof Ivo Tews – University of Southampton

Dr Anastasios Tsaousis – University of Kent

Summary

The major protein transport routes into chloroplasts are thought to be well understood. However, new discoveries challenge current textbook models.

We have recently identified a novel protein translocator in red algae. The so far unannotated gene, which encodes for a membrane OMP85 family protein, is unusually encoded directly in the chloroplast genome. This localization is an intermediate evolutionary state that could explain how protein translocators in plant chloroplasts once evolved to import proteins from an ancestral protein export role in cyanobacteria.

The aims of the project are therefore to investigate whether the new translocator can transport protein substrates into the chloroplast and whether its substrate-recognition domain is oriented outwards from the embedding membrane as in plant chloroplasts or inwards as in cyanobacteria. To ascertain the directionality and function of the translocator, a three-pronged approach will be used, which combines comparative genomics (Janouskovec), cell biology and biochemistry (Tsaousis), and structural biology techniques (Tews) contributed by the three hosting laboratories.

You will use:

• microscopy and molecular cell biology to localise the translocator in the chloroplast membrane, determine its orientation by immunolabeling, and test its functionality by conditional gene knockout/knockin in algal models Porphyridium or Cyanidioschyzon;

• biochemistry and crystallography to study the 3D structure of the membrane translocator and understand the mechanics of protein transport through studying its cargo recognition POTRA domains;

• bioinformatics to determine the structure, distribution and phylogeny of the translocator and use this to update models on how chloroplasts became integrated into eukaryotic cells.

The findings will reshape our views on how plants and algae became photosynthetic and underwent massive reduction in their chloroplast genomes by acquiring the ability to import nucleus-encoded proteins.