Substrate Recognition by the Yeast Maltose Transporter 

Master’s Research Project
Group: Membrane enzymology
Supervisors: Ryan K. Henderson
Prof. Dr. Bert Poolman

Introduction

The biological membrane is a remarkable feature of the cell that acts as a barrier against the outside environment. However, cells require nutrients from their surroundings, so the membrane must not be a perfect barrier. Thus, proteins able to bridge the membrane and transport molecules are absolutely essential to life, and are found in any membrane in nature. The largest and most diverse family of membrane transport proteins is the Major Facilitator Superfamily (MFS), which is found across all domains of life. While MFS transporters are structurally similar, they can transport a wide variety of crucial biological molecules including sugars, ions, peptides, vitamins, and drugs.
The maltose permease Mal11 from budding yeast Saccharomyces cerevisiae is one such MFS transporter. Mal11 acts as the first step of yeast maltose metabolism by transporting maltose (coupled to a proton) across the plasma membrane and into the cell. Mal11 is one of several yeast maltose transporters, but is distinct from the others in that it has quite a broad range of specificity for related sugars. While this protein shares significant similarity with other transporters, the molecular basis of this “substrate promiscuity” remains elusive, as are the structural rearrangements necessary for Mal11 to carry out transport.

Project description

This project aims to characterize some of the fundamental molecular interactions governing Mal11 structure and function. Amino acid residues of interest have previously been identified based on a structural model of Mal11, and single amino acid mutations have been made to these residues. In this project, these mutants will be tested for their kinetic activity, substrate specificity, and energy coupling mechanisms. Using the information gleaned from this characterization, additional mutants and combinations of mutants can be examined to pinpoint critical interactions within Mal11 and between Mal11 and its substrates. Besides the scientific knowledge gained, this project has industrial relevance too. Yeast is extensively used in the production of ethanol and other chemicals, and maltose is an important carbon source used in such settings.
Techniques that will be taught and used include: yeast and bacterial genetics, culturing, and cloning; protein engineering and making of mutants; radiolabeled and fluorescence-based transport measurements; protein structure analysis.

ATP Sensing in Yeast 

Bachelor Research Project
Group: Membrane enzymology
Supervisors: Ryan K. Henderson
Bauke F. Gaastra
Prof. Dr. Bert Poolman

Background

The biological membrane is a remarkable feature of the cell that acts as a barrier against the outside environment. However, cells require nutrients from their surroundings, so the membrane must not be a perfect barrier. Thus, proteins able to bridge the membrane and transport molecules are absolutely essential to life, and are found in any membrane in nature. One of the most common ways a transporter can couple solute movement to an energy gradient is secondary active transport, which is used for a wide variety of crucial biological molecules including sugars, ions, peptides, vitamins, and drugs.
Yeast (Saccharomyces cerevisiae) is a single-celled eukaryote that is used extensively in basic scientific research and in industrial processes. An important aspect for industrial yeast strains is to use energy (i.e: ATP) very efficiently. One way to do this is to make solute transport more efficient. In our lab, we’re interested in the secondary active transporter Mal11, a maltose-proton symporter.

Project description

This project aims to introduce an ATP sensor into yeast and use it to develop a useful and robust fluorescence-based assay for ATP production or consumption. Once this assay is verified, we will use the assay to assess whether mutations to Mal11 can help to conserve ATP. Techniques that will be taught and used include: yeast and bacterial genetics, culturing, and cloning; assay development; fluorescence-based measurements.

Biophysical characterization of a structural protein involved in cell volume regulation 

Group: Membrane enzymology
Supervisors: Prof. Bert Poolman
Daily supervisor: Dr. Aditya Iyer

Background

Bacteria are constantly exposed to osmotic environments that pose a danger to their survival. The cytoplasm of bacteria is significantly more concentrated than the extracellular environment producing a positive outward pressure called turgor. At severe osmotic upshifts (hyperosmotic stress), the difference between turgor and external pressure approaches zero resulting in deflation of the cell (plasmolysis)1. This is because osmotic upshifts cause water movement out of the cell. Conversely, at severe osmotic downshifts, water will inflate the cell and may cause cell lysis. Cell survival under osmotic stress conditions crucially relies on regulatory mechanisms to maintain volume homeostasis2. Existing osmoadaptive mechanisms are incomplete as they rely on the fact that the availability of osmolyte(s), rate of their transport and synthesis of osmo-protectants are not limiting; an unlikely situation since ion/solute concentrations in diverse environments differ spatiotemporally. Mechanism(s) that help(s) in osmoadaptation/osmotolerance independent of osmolyte or metabolic energy availability would aid bacterial survival. The immediate consequence of osmotic upshift to E. coli is shrinking of the cytoplasmic-volume (plasmolysis) and possible loss of the cytoplasmic structure (Fig. 1). Interestingly in plasmolysing cells, the inner membrane (IM) remains attached to the outer membrane (OM) in certain regions suggesting that the cytoplasm of E. coli is somehow prevented from collapsing completely. We propose a regulatory mechanism wherein the inner and outer membrane are physically held together by an osmotically-inducible protein Y (OsmY); this protein is overexpressed under hyperosmotic stress (Fig. 1).

Osmos
Figure 1: Hypothesis for osmoprotective role of the OsmY (blue) protein in E.coli.
The cytoplasm is shown in yellow and the nucleoid in green.
The protein OsmY is proposed to link the inner and outer membrane, and hence control the cellular volume3. But the secondary structure of OsmY and the details of OsmY-lipid membrane interaction have not been investigated yet.

Project description

In this project, you will purify OsmY and carry out for the first time a comprehensive biophysical characterization of the protein. The secondary structure of OsmY will be investigated using circular dichroism (CD) spectroscopy and light scattering techniques. Additionally, phospholipid membrane binding studies will be carried out using isothermal calorimetry (ITC)4. If time permits, you will learn how to engineer single point mutations in the OsmY for fluorophore labeling or modification of the (e.g. membrane-binding) properties of the protein. Your project is a part of ongoing studies in the lab pertaining to regulatory mechanisms underlying survival of cells under (extreme) osmotic stress.

Outline of the project

Part I – Molecular biology and biochemistry
  •  Isolation of OsmY gene from E. coli using polymerase chain reaction (PCR)
  • Creating plasmids for inducible expression of OsmY
  • Transformation of plasmids to E. coli
  • Large-scale expression and His-tag purification of OsmY
  • Validation of purified protein using established techniques
  Part II – Characterization of OsmY protein
  • CD spectroscopy of OsmY protein under varying ionic strengths and osmolarities, and probe how binding to phospholipid membranes binding affects the secondary structure
  • Fluorescence experiments to probe aggregation and surface properties using polarity-sensitive probes like ANS, FE etc.
  • ITC measurements to probe phospholipid membrane binding
  • Localization studies in live cells using fluorescent-tagged OsmY

References

1. Pilizota, T. & Shaevitz, J. W. Origins of escherichia coli growth rate and cell shape changes at high external osmolality. Biophys. J. 107, 1962–1969 (2014).
2. van den Berg J, Boersma AJ & Poolman B (2017) Bacterial cells maintain crowding homeostasis. Nature Rev Microbiol, in press.
3. Liechty, A., Chen, J. & Jain, M. K. Origin of antibacterial stasis by polymyxin B in Escherichia coli. Biochim. Biophys. Acta – Biomembr. 1463, 55–64 (2000).
4. Du, X. et al. Insights into Protein-Ligand Interactions: Mechanisms, Models, and Methods. Int. J. Mol. Sci. 17, 144 (2016).