Ewald Hettema - Ewald Hettema - People - Molecular Biology and Biotechnology

Dr Ewald Hettema

Reader in Molecular Cell Biology

Tel: 0114 222 2732
Email: e.hettema@sheffield.ac.uk

Research	Research Precis My group aims to improve understanding of the molecular mechanisms underlying peroxisome dynamics at the cellular level. Cells contain a large number of distinct membrane bound organelles. These compartments rely on complex machineries to acquire and maintain their unique composition and function. We are studying one of these organelles, the peroxisome. Lack of functional peroxisomes results in a deficiency of a large number of enzymatic reactions and a disorder called Zellweger (ZS-) or cerebro-hepato-renal syndrome. Proteins required for peroxisome formation are called peroxins and these have been found mutated in ZS patients. Peroxisomes multiply by growth and division. The endoplasmic reticulum (ER) provides peroxisomes with membrane constituents allowing these organelles to grow, after which the organelles divide and distribute between mother and daughter cell. The number of peroxisomes per cell is influenced by intracellular and extracellular factors and these can induce proliferation or breakdown. How these processes are regulated and integrated into cellular metabolism is poorly understood. Fig 2A. Peroxisomes multiply and are subsequently transported to the newly forming daughter cell via an actin-myosin-based process. Approximately half the number of the peroxisomes are anchored to the periphery of mother cells and remain there. These opposing processes ensure peroxisome segregation with high fidelity. Fig 2B. Peroxisomes can be induced to proliferate under conditions of high requirement for these organelles. A subsequent shift to conditions where peroxisomes are superfluous induces their rapid breakdown by autophagy. Selective breakdown of peroxisomes is named pexophagy. Genetic model organisms to study peroxisome dynamics and functioning We and others have been using Saccharomyces cerevisiae as our model system and mutants have been identified that are disturbed in various aspects of peroxisome dynamics. These mutants have been instrumental in unravelling the underlying mechanisms of peroxisome formation, multiplication and segregation. We have recently focussed on the peroxisomal membrane protein Pex3. Lack of Pex3 results in a complete absence of peroxisomal structures. We have now found that Pex3 is also involved in peroxisome segregation during cell division and peroxisome turnover. It does this by binding of process-specific factors and therefore may act as a scaffold for the regulation of peroxisome dynamics. Fig 3 shows. Mutants in S.cerevisiae cells are gene deletion mutants. Mutant phenotypes in D. melanogaster S2 cells were created by temporary inactivation of genes with RNAi. Blue circumference of yeast cells is artificially coloured bright field image. Nuclei are stained with DAPI in S2 cells and artificially coloured blue in images. More recently we have extended our studies into other model organisms including the fruit fly Drosophila melanogaster, the slime mould Dictyostelium discoideum and human cells. For instance, a genome-wide RNAi screen in Drosophila cells identified many of the known proteins involved in peroxisome formation and peroxisome multiplication. Interestingly, we also identified several new genes that appear to be involved in formation and regulation of peroxisome dynamics. We use molecular cell biological approaches including live-cell imaging, genome-wide RNAi screens, yeast genetics, proteomics and protein-protein interaction studies to characterise the function of these new proteins. Although our main interest is in a fundamental understanding of peroxisome dynamics and its role in peroxisome functioning, we are also following up on lines of research that potentially have medical implications. Research Keywords Eukaryotic cell biology, yeast genetics, Saccharomyces cerevisiae, peroxisomes, autophagy, peroxisome biogenesis disorders I welcome applications from self-funded prospective home and international PhD students; see examples of possible projects below. You can apply for a PhD position in MBB here. Contact me at e.hettema@sheffield.ac.uk for further information. Examples of self-funded PhD projects: Development of genome editing tools for use in non-model organisms with biotechnological potential Construction of synthetic organelles for biotechnology Investigations into the molecular mechanisms of organelle movement and inheritance Characterisation of peroxisome fission and formation Fungal extracellular vesicle formation
Teaching	Level 3 Modules MBB342 Genetics of Cell Growth and Division Level 1 Modules MBB164 Molecular Biology
Career History	Career History 2014 - present: Reader in Molecular Cell Biology, Dept. Molecular Biology and Biotechnology, University of Sheffield 2008 - 2014: Wellcome Trust Senior Research Fellow in Basic Biomedical Science at University of Sheffield 2004 - 2008: Wellcome Trust Career Development Fellow at University of Sheffield 2000 - 2004: Postdoctoral Reserach Associate, MRC LMB, Cambridge 1998 - 2000: Postdoctoral Reserach Associate, University of Amsterdam 1998: PhD, University of Amsterdam

Research

Research Precis

fig1 My group aims to improve understanding of the molecular mechanisms underlying peroxisome dynamics at the cellular level.

Cells contain a large number of distinct membrane bound organelles. These compartments rely on complex machineries to acquire and maintain their unique composition and function. We are studying one of these organelles, the peroxisome. Lack of functional peroxisomes results in a deficiency of a large number of enzymatic reactions and a disorder called Zellweger (ZS-) or cerebro-hepato-renal syndrome. Proteins required for peroxisome formation are called peroxins and these have been found mutated in ZS patients.

Peroxisomes multiply by growth and division.

The endoplasmic reticulum (ER) provides peroxisomes with membrane constituents allowing these organelles to grow, after which the organelles divide and distribute between mother and daughter cell. The number of peroxisomes per cell is influenced by intracellular and extracellular factors and these can induce proliferation or breakdown. How these processes are regulated and integrated into cellular metabolism is poorly understood.

fig2 Fig 2A. Peroxisomes multiply and are subsequently transported to the newly forming daughter cell via an actin-myosin-based process. Approximately half the number of the peroxisomes are anchored to the periphery of mother cells and remain there. These opposing processes ensure peroxisome segregation with high fidelity. Fig 2B. Peroxisomes can be induced to proliferate under conditions of high requirement for these organelles. A subsequent shift to conditions where peroxisomes are superfluous induces their rapid breakdown by autophagy. Selective breakdown of peroxisomes is named pexophagy.

Genetic model organisms to study peroxisome dynamics and functioning

We and others have been using Saccharomyces cerevisiae as our model system and mutants have been identified that are disturbed in various aspects of peroxisome dynamics. These mutants have been instrumental in unravelling the underlying mechanisms of peroxisome formation, multiplication and segregation. We have recently focussed on the peroxisomal membrane protein Pex3. Lack of Pex3 results in a complete absence of peroxisomal structures. We have now found that Pex3 is also involved in peroxisome segregation during cell division and peroxisome turnover. It does this by binding of process-specific factors and therefore may act as a scaffold for the regulation of peroxisome dynamics.

fig3

Fig 3 shows. Mutants in S.cerevisiae cells are gene deletion mutants. Mutant phenotypes in D. melanogaster S2 cells were created by temporary inactivation of genes with RNAi. Blue circumference of yeast cells is artificially coloured bright field image. Nuclei are stained with DAPI in S2 cells and artificially coloured blue in images.

More recently we have extended our studies into other model organisms including the fruit fly Drosophila melanogaster, the slime mould Dictyostelium discoideum and human cells. For instance, a genome-wide RNAi screen in Drosophila cells identified many of the known proteins involved in peroxisome formation and peroxisome multiplication. Interestingly, we also identified several new genes that appear to be involved in formation and regulation of peroxisome dynamics. We use molecular cell biological approaches including live-cell imaging, genome-wide RNAi screens, yeast genetics, proteomics and protein-protein interaction studies to characterise the function of these new proteins.

Although our main interest is in a fundamental understanding of peroxisome dynamics and its role in peroxisome functioning, we are also following up on lines of research that potentially have medical implications.

Research Keywords

Eukaryotic cell biology, yeast genetics, Saccharomyces cerevisiae, peroxisomes, autophagy, peroxisome biogenesis disorders

I welcome applications from self-funded prospective home and international PhD students; see examples of possible projects below. You can apply for a PhD position in MBB here.

Contact me at e.hettema@sheffield.ac.uk for further information.

Examples of self-funded PhD projects:

Development of genome editing tools for use in non-model organisms with biotechnological potential

Construction of synthetic organelles for biotechnology

Investigations into the molecular mechanisms of organelle movement and inheritance

Characterisation of peroxisome fission and formation

Fungal extracellular vesicle formation

Teaching

Level 3 Modules

MBB342 Genetics of Cell Growth and Division

Level 1 Modules

MBB164 Molecular Biology

Career History

            2014 - present: Reader in Molecular Cell Biology, Dept. Molecular Biology and Biotechnology, University of Sheffield
- 2014: Wellcome Trust Senior Research Fellow in Basic Biomedical Science at University of Sheffield
- 2008: Wellcome Trust Career Development Fellow at University of Sheffield
- 2004: Postdoctoral Reserach Associate, MRC LMB, Cambridge
- 2000: Postdoctoral Reserach Associate, University of Amsterdam
PhD, University of Amsterdam

            

Publications

Journal articles

Devarajan S, Meurer M, van Roermund CWT, Chen X, Hettema EH, Kemp S, Knop M & Williams C (2020) Proteasome-dependent protein quality control of the peroxisomal membrane protein Pxa1p. Biochimica et Biophysica Acta (BBA) - Biomembranes.
Al-Saryi NA, Al-Hejjaj MY, van Roermund CWT, Hulmes GE, Ekal L, Payton C, Wanders RJA & Hettema EH (2017) Two NAD-linked redox shuttles maintain the peroxisomal redox balance in Saccharomyces cerevisiae. Scientific Reports, 7(1). View this article in WRRO
Saryi NA, Hutchinson JD, Al-Hejjaj MY, Sedelnikova S, Baker P & Hettema EH (2017) Pnc1 piggy-back import into peroxisomes relies on Gpd1 homodimerisation.. Scientific Reports, 7. View this article in WRRO
Hettema EH & Gould SJ (2017) Cell biology: Organelle formation from scratch. Nature, 542, 174-175.
Motley AM, Galvin PC, Ekal L, Nuttall JM & Hettema EH (2015) Reevaluation of the role of Pex1 and dynamin-related proteins in peroxisome membrane biogenesis. The Journal of Cell Biology, 211(5), 1041-1056. View this article in WRRO
Kim PK & Hettema EH (2015) Multiple Pathways for Protein Transport to Peroxisomes. Journal of Molecular Biology, 427(6), 1176-1190. View this article in WRRO
Hettema EH, Erdmann R, van der Klei I & Veenhuis M (2014) Evolving models for peroxisome biogenesis. Current Opinion in Cell Biology, 29(1), 25-30. View this article in WRRO
Nuttall JM, Motley AM & Hettema EH (2014) Deficiency of the exportomer components Pex1, Pex6, and Pex15 causes enhanced pexophagy in Saccharomyces cerevisiae.. Autophagy, 10(5), 835-845. View this article in WRRO
Fakieh MH, Drake PJM, Lacey J, Munck JM, Motley AM & Hettema EH (2013) Intra-ER sorting of the peroxisomal membrane protein Pex3 relies on its luminal domain.. Biol Open, 2(8), 829-837. View this article in WRRO
Nuttall J, Motley A & Hettema E (2013) Pexophagy is activated in a subset of peroxisome assembly mutants. MOLECULAR BIOLOGY OF THE CELL, 24.
Nuttall JM, Hettema EH & Watts DJ (2012) Farnesyl diphosphate synthase, the target for nitrogen-containing bisphosphonate drugs, is a peroxisomal enzyme in the model system Dictyostelium discoideum.. Biochem J, 447(3), 353-361. View this article in WRRO
Motley AM, Nuttall JM & Hettema EH (2012) Atg36: the Saccharomyces cerevisiae receptor for pexophagy.. Autophagy, 8(11), 1680-1681.
Motley AM, Nuttall JM & Hettema EH (2012) Pex3-anchored Atg36 tags peroxisomes for degradation in Saccharomyces cerevisiae.. EMBO J, 31(13), 2852-2868. View this article in WRRO
Nuttall JM, Motley A & Hettema EH (2011) Peroxisome biogenesis: Recent advances. Current Opinion in Cell Biology, 23(4), 421-426.
Smaczynska-de Rooij II, Allwood EG, Aghamohammadzadeh S, Hettema EH, Goldberg MW & Ayscough KR (2010) A role for the dynamin-like protein Vps1 during endocytosis in yeast.. J Cell Sci, 123(Pt 20), 3496-3506.
Munck JM, Motley AM, Nuttall JM & Hettema EH (2009) A dual function for Pex3p in peroxisome formation and inheritance.. J Cell Biol, 187(4), 463-471. View this article in WRRO
Hettema EH & Motley AM (2009) How peroxisomes multiply.. J Cell Sci, 122(Pt 14), 2331-2336.
Motley AM, Ward GP & Hettema EH (2008) Dnm1p-dependent peroxisome fission requires Caf4p, Mdv1p and Fis1p.. J Cell Sci, 121(Pt 10), 1633-1640.
Motley AM & Hettema EH (2007) Yeast peroxisomes multiply by growth and division.. J Cell Biol, 178(3), 399-410. View this article in WRRO
Hettema EH & Ayscough KR (2007) Immunological methods. METHOD MICROBIOL, 36, 241-268.
Hettema EH, Valdez-Taubas J & Pelham HRB (2004) Bsd2 binds the ubiquitin ligase Rsp5 and mediates the ubiquitination of transmembrane proteins.. EMBO J, 23(6), 1279-1288.
Hettema EH, Lewis MJ, Black MW & Pelham HRB (2003) Retromer and the sorting nexins Snx4/41/42 mediate distinct retrieval pathways from yeast endosomes.. EMBO J, 22(3), 548-557.
Hoepfner D, van den Berg M, Philippsen P, Tabak HF & Hettema EH (2001) A role for Vps1p, actin, and the Myo2p motor in peroxisome abundance and inheritance in Saccharomyces cerevisiae.. J Cell Biol, 155(6), 979-990. View this article in WRRO
van Roermund CW, Drissen R, van Den Berg M, Ijlst L, Hettema EH, Tabak HF, Waterham HR & Wanders RJ (2001) Identification of a peroxisomal ATP carrier required for medium-chain fatty acid beta-oxidation and normal peroxisome proliferation in Saccharomyces cerevisiae.. Mol Cell Biol, 21(13), 4321-4329.
van Roermund CWT, van den Berg M, Tabak HF, Wanders RJA & Hettema EH (2001) Peroxisomal beta-oxidation in yeast: new insights into the mechanisms involved with emphasis on the role of PEX11P and YPR128CP, two peroxisomal membrane proteins, in betaoxidation and peroxisomal proliferation. Biochemical Society Transactions, 29(1), A29-A29.
van Roermund CW, Tabak HF, van Den Berg M, Wanders RJ & Hettema EH (2000) Pex11p plays a primary role in medium-chain fatty acid oxidation, a process that affects peroxisome number and size in Saccharomyces cerevisiae.. J Cell Biol, 150(3), 489-498. View this article in WRRO
Motley AM, Hettema EH, Ketting R, Plasterk R & Tabak HF (2000) Caenorhabditis elegans has a single pathway to target matrix proteins to peroxisomes.. EMBO Rep, 1(1), 40-46.
Hettema EH & Tabak HF (2000) Transport of fatty acids and metabolites across the peroxisomal membrane.. Biochim Biophys Acta, 1486(1), 18-27.
Barnett P, Tabak HF & Hettema EH (2000) Nuclear receptors arose from pre-existing protein modules during evolution.. Trends Biochem Sci, 25(5), 227-228.
Hettema EH, Girzalsky W, van Den Berg M, Erdmann R & Distel B (2000) Saccharomyces cerevisiae pex3p and pex19p are required for proper localization and stability of peroxisomal membrane proteins.. EMBO J, 19(2), 223-233.
Kal AJ, Hettema EH, van den Berg M, Koerkamp MG, van Ijlst L, Distel B & Tabak HF (2000) In silicio search for genes encoding peroxisomal proteins in Saccharomyces cerevisiae.. Cell Biochem Biophys, 32 Spring, 1-8.
van Roermund CW, Hettema EH, van den Berg M, Tabak HF & Wanders RJ (1999) Molecular characterization of carnitine-dependent transport of acetyl-CoA from peroxisomes to mitochondria in Saccharomyces cerevisiae and identification of a plasma membrane carnitine transporter, Agp2p.. EMBO J, 18(21), 5843-5852.
Hettema EH, Distel B & Tabak HF (1999) Import of proteins into peroxisomes.. Biochim Biophys Acta, 1451(1), 17-34.
Stroobants AK, Hettema EH, van den Berg M & Tabak HF (1999) Enlargement of the endoplasmic reticulum membrane in Saccharomyces cerevisiae is not necessarily linked to the unfolded protein response via Ire1p.. FEBS Lett, 453(1-2), 210-214.
Hettema EH, Ruigrok CC, Koerkamp MG, van den Berg M, Tabak HF, Distel B & Braakman I (1998) The cytosolic DnaJ-like protein djp1p is involved specifically in peroxisomal protein import.. J Cell Biol, 142(2), 421-434. View this article in WRRO
Brites P, Motley A, Hogenhout E, Hettema E, Wijburg F, Heijmans HS, Tabak HF, Distel B & Wanders RJ (1998) Molecular basis of rhizomelic chondrodysplasia punctata type I: high frequency of the Leu-292 stop mutation in 38 patients.. J Inherit Metab Dis, 21(3), 306-308.
Ofman R, Hettema EH, Hogenhout EM, Caruso U, Muijsers AO & Wanders RJ (1998) Acyl-CoA:dihydroxyacetonephosphate acyltransferase: cloning of the human cDNA and resolution of the molecular basis in rhizomelic chondrodysplasia punctata type 2.. Hum Mol Genet, 7(5), 847-853.
van Roermund CW, Hettema EH, Kal AJ, van den Berg M, Tabak HF & Wanders RJ (1998) Peroxisomal beta-oxidation of polyunsaturated fatty acids in Saccharomyces cerevisiae: isocitrate dehydrogenase provides NADPH for reduction of double bonds at even positions.. EMBO J, 17(3), 677-687.
Verleur N, Hettema EH, van Roermund CW, Tabak HF & Wanders RJ (1997) Transport of activated fatty acids by the peroxisomal ATP-binding-cassette transporter Pxa2 in a semi-intact yeast cell system.. Eur J Biochem, 249(3), 657-661.
Motley AM, Hettema EH, Hogenhout EM, Brites P, ten Asbroek AL, Wijburg FA, Baas F, Heijmans HS, Tabak HF, Wanders RJ & Distel B (1997) Rhizomelic chondrodysplasia punctata is a peroxisomal protein targeting disease caused by a non-functional PTS2 receptor.. Nat Genet, 15(4), 377-380.
Hettema EH, van Roermund CW, Distel B, van den Berg M, Vilela C, Rodrigues-Pousada C, Wanders RJ & Tabak HF (1996) The ABC transporter proteins Pat1 and Pat2 are required for import of long-chain fatty acids into peroxisomes of Saccharomyces cerevisiae.. EMBO J, 15(15), 3813-3822.
Tabak HF, Elgersma Y, Hettema E, Franse MM, Voorn-Brouwer T & Distel B (1995) Transport of proteins and metabolites across the impermeable membrane of peroxisomes.. Cold Spring Harb Symp Quant Biol, 60, 649-655.
Légaré D, Hettema E & Ouellette M (1994) The P-glycoprotein-related gene family in Leishmania.. Mol Biochem Parasitol, 68(1), 81-91.
Motley A, Hettema E, Distel B & Tabak H (1994) Differential protein import deficiencies in human peroxisome assembly disorders.. J Cell Biol, 125(4), 755-767. View this article in WRRO
Bank RA, Hettema EH, Muijs MA, Pals G, Arwert F, Boomsma DI & Pronk JC (1992) Variation in gene copy number and polymorphism of the human salivary amylase isoenzyme system in Caucasians.. Hum Genet, 89(2), 213-222.
Ouellette M, Hettema E, Wüst D, Fase-Fowler F & Borst P (1991) Direct and inverted DNA repeats associated with P-glycoprotein gene amplification in drug resistant Leishmania.. EMBO J, 10(4), 1009-1016.
Bank RA, Hettema EH, Arwert F, Amerongen AV & Pronk JC (1991) Electrophoretic characterization of posttranslational modifications of human parotid salivary alpha-amylase.. Electrophoresis, 12(1), 74-79.

Conference proceedings papers

Al-hejjaj M, Watts D & Hettema EH (2016) Attempt to develop a CRISPR system for use in Dictyostelium discoideum.. MOLECULAR BIOLOGY OF THE CELL, Vol. 27