Advanced

Effects of phosphorylation on oligomerisation and localisation of the Streptomyces coelicolor protein DivIVA in vitro

Miksys, Algirdas (2016) MOBT01 20152
Degree Projects in Molecular Biology
Abstract
Streptomyces is the largest genus in the phylum Actinobacteria, known for their complex life cycle and secondary metabolite production. The model bacterium Streptomyces coelicolor grows _lamentously by tip extension and hyphal branching. The hyphal tip is highly organised, with various structural proteins forming a large assembly termed the polarisome. An essential protein, DivIVA, localises to the tips of the hyphae and has been shown to mark new branching sites. DivIVA has homologues in many Grampositive bacteria, and was shown to be essential for viability and direct polar growthin tested mycobacteria, corynebacteria and streptomycetes. DivIVA is always found at cell poles, but it is unclear how it is targeted to this subcellular... (More)
Streptomyces is the largest genus in the phylum Actinobacteria, known for their complex life cycle and secondary metabolite production. The model bacterium Streptomyces coelicolor grows _lamentously by tip extension and hyphal branching. The hyphal tip is highly organised, with various structural proteins forming a large assembly termed the polarisome. An essential protein, DivIVA, localises to the tips of the hyphae and has been shown to mark new branching sites. DivIVA has homologues in many Grampositive bacteria, and was shown to be essential for viability and direct polar growthin tested mycobacteria, corynebacteria and streptomycetes. DivIVA is always found at cell poles, but it is unclear how it is targeted to this subcellular location. Studies on B. subtilis DivIVA have proposed that it localises by sensing negative membrane curvature. Recently, it has been shown that S. coelicolor DivIVA is phosphorylated by the serine/threonine protein kinase AfsK. Overexpression of AfsK has dramatic e_ects on hyphal branching and DivIVA localisation in vivo. In this study, we try to set-up an experimental system to investigate the e_ects of phosphorylation on DivIVA and how it a_ects assembly of the protein on membrane surfaces in vitro. Native, phosphomimic and phosphorylated proteins were puri_ed and investigated by native gel electrophoresis and size exclusion chromatography. Fluorescent protein tagged versions were used for supported lipid bilayer binding experiments. We show that phosphorylation does not seem to have dramatic e_ects on oligomeric states of DivIVA, and that bilayer lipid composition is important in membrane binding. (Less)
Popular Abstract
DivIVA likes curviness and cooperation

The Earth is round. But it feels flat. And if you lay down with your arms outstretched, it doesn’t feel like lying on a ball.

A similar problem is facing a protein of the bacterium Streptomyces coelicolor. This bacterium grows in long, branching threads – hyphae – and the protein in question, DivIVA (Div-four-A) is found at the very tips of every filament. It also seems to be the main player in establishing new branches along the side walls of the hyphae. How does it localize to the cell tip?

DivIVA binds to the membrane inside the cell, is present in large numbers at the cell tip, and forms big complexes. It can become modified by another protein, which attaches phosphate groups to DivIVA –... (More)
DivIVA likes curviness and cooperation

The Earth is round. But it feels flat. And if you lay down with your arms outstretched, it doesn’t feel like lying on a ball.

A similar problem is facing a protein of the bacterium Streptomyces coelicolor. This bacterium grows in long, branching threads – hyphae – and the protein in question, DivIVA (Div-four-A) is found at the very tips of every filament. It also seems to be the main player in establishing new branches along the side walls of the hyphae. How does it localize to the cell tip?

DivIVA binds to the membrane inside the cell, is present in large numbers at the cell tip, and forms big complexes. It can become modified by another protein, which attaches phosphate groups to DivIVA – it becomes phosphorylated. When that happens, DivIVA delocalizes from the tips of the hyphae, and after some time many new branches are formed along the side walls.

A very interesting feature that has been shown in studies with proteins similar to DivIVA in other bacteria is that they localize to the cell tips by sensing the curvature of the membrane. The size of the protein is about 2 nm, and the cell is about 1000 nm wide, so it is like trying to measure a 750 m wide ball by trying to wrap your arms around it. It seems impossible, but if the protein forms large complexes, it might stand a chance.

We set out to see how large these complexes are and if phosphorylation has any effect on their size. We did this by purifying DivIVA, both unmodified and phosphorylated, and also a mutant protein that mimics phosphorylation, and testing each of them by separating them by size using an electric field (electrophoresis), or a column which separates molecules by their size (Size Exclusion Chromatography). We found no difference between the variants – all of them formed large complexes made up of tens of proteins.

We then tried to see if we can make it bind to model membranes made in the laboratory. For that, we tagged DivIVA with a fluorescent protein, and tried to see if it binds to membranes assembled on glass slides. We found that they seem to do, but it is dependent on membrane composition – the components of the membrane that were more bacteria-like seemed to bind it much better. Knowing that, and after ironing out a few kinks, the next, most exciting, steps would be to see how DivIVA binds to membranes that are curved.

Advisor: Klas Flärdh
Master´s Degree Project of 60 credits in Molecular Biology, Microbiology 2015-2016
Department of Biology, Lund University (Less)
Please use this url to cite or link to this publication:
author
Miksys, Algirdas
supervisor
organization
course
MOBT01 20152
year
type
H2 - Master's Degree (Two Years)
subject
language
English
id
8894694
date added to LUP
2016-11-07 15:17:50
date last changed
2016-11-07 15:17:50
@misc{8894694,
  abstract     = {Streptomyces is the largest genus in the phylum Actinobacteria, known for their complex life cycle and secondary metabolite production. The model bacterium Streptomyces coelicolor grows _lamentously by tip extension and hyphal branching. The hyphal tip is highly organised, with various structural proteins forming a large assembly termed the polarisome. An essential protein, DivIVA, localises to the tips of the hyphae and has been shown to mark new branching sites. DivIVA has homologues in many Grampositive bacteria, and was shown to be essential for viability and direct polar growthin tested mycobacteria, corynebacteria and streptomycetes. DivIVA is always found at cell poles, but it is unclear how it is targeted to this subcellular location. Studies on B. subtilis DivIVA have proposed that it localises by sensing negative membrane curvature. Recently, it has been shown that S. coelicolor DivIVA is phosphorylated by the serine/threonine protein kinase AfsK. Overexpression of AfsK has dramatic e_ects on hyphal branching and DivIVA localisation in vivo. In this study, we try to set-up an experimental system to investigate the e_ects of phosphorylation on DivIVA and how it a_ects assembly of the protein on membrane surfaces in vitro. Native, phosphomimic and phosphorylated proteins were puri_ed and investigated by native gel electrophoresis and size exclusion chromatography. Fluorescent protein tagged versions were used for supported lipid bilayer binding experiments. We show that phosphorylation does not seem to have dramatic e_ects on oligomeric states of DivIVA, and that bilayer lipid composition is important in membrane binding.},
  author       = {Miksys, Algirdas},
  language     = {eng},
  note         = {Student Paper},
  title        = {Effects of phosphorylation on oligomerisation and localisation of the Streptomyces coelicolor protein DivIVA in vitro},
  year         = {2016},
}