The yeast colony color sectoring screen

Colony-color sectoring

• Classical experiment by D. Koshland, John Kent and Lee Hartwell

• Genetic analysis of mitotic transmission of minichromosomes (CEN/ARS vector)

• Used ade2, ade3 double mutant and centromeric vector carrying the ADE3 gene

ade2

ade2

ade3

ade3

ade3

ade3ade2

ade2

ADE3

CEN

3

ARS1

LEU

2

ADE3

CEN

3

ARS1

LEU

2

Also Leu-

Mutagenize – look for mutations that affect plasmid maintenance

ade2

ade2

ade3

ade3

ADE3

CEN

3ARS1

LEU

2

ade2

ade2

ade3

ade3

ADE3

CEN

3

ARS1

LEU

2

ade2

ade2

ade3

ade3

ADE3

CEN

3

ARS1

LEU

2

ade2

ade2

ade3

ade3

ADE3

CEN

3

ARS1

LEU

2

ade2

ade2

ade3

ade3

ADE3

CEN

3

ARS1

LEU

2

ade2

ade2

ade3

ade3

ADE3

CEN

3

ARS1

LEU

2

ade2

ade2

ade3

ade3

ADE3

CEN

3

ARS1

LEU

2

ade3

ade3ade2

ade2

sectored colonie

ade2

ade2

ade3

ade3

ade3

ade3ade2

ade2

ADE3

CEN

3

GENEX

LEU

2

ADE3

CEN

3

GENEX

LEU

2

Also Leu-

Mutagenize – generate mutations that will make cell survival dependent on presence of the plasmid

ade2

ade2

ade3

ade3

ADE3

CEN

3

GENEX

LEU

2

ade2

ade2

ade3

ade3

ADE3

CEN

3

GENEX

LEU

2

ade2

ade2

ade3

ade3

ADE3

CEN

3

GENEX

LEU

2

What other uses for colony color sectoring screen?

• Cloning by function – Identification of factors with functions that

cannot be found by homology searcher• Synthetic lethal screens

– Identification of genetic interactions of your favorite gene

NON-sectored colonie

Cloning by function

A real-life screen: search for a mitochondrial fatty acyl-ACP dehydratase

• Mitochondrial fatty acid synthesis: relatively newly discovered pathway

• Essential for respiration in yeast• Conserved in humans• Human homologues can complement

yeast deletion mutants when expressed in yeast functional conservation

Acetyl-CoA Carboxylase

(ACC1), HFA1 ?

Malonyl-CoA-ACP Transacylase (MCT1)

Acyl carrier protein (ACP1)

Acetyl-CoA-ACP-transacylase

O

C

O

ACP

3-ketoacyl-ACP synthase (CEM1)

Acetyl-CoA

?

(type II FAS)

fabD

acpPfabH

fabF

Ketoacyl-ACP Reductase (OAR1)

Hydroxyacyl-ACP Dehydratase ?

trans-2-enoyl-ACP reductase (MRF1)

3-ketoacyl-ACP synthase (CEM1)

C

O

ACPO

-O

CO2, ACPSH

+ ACPSH

C

O

O-

thioesterase?

fabG

fabAfabZ

fabI

fabF

Phenotypes of strains deleted for components of the mitochondrial FAS

• respiratory deficient pet phenotype• loss of spectrally dectectable mitochondrial

cytochromes• loss of mitochondrial DNA in some cases

(Δacp1, Δppt2)• no marked change in fatty acid composition• lack of lipoic acid, a cofactor essential for

function of a-ketglutarate dehydrogenase and pyruvate dehydrogenase

- A deletion mutation of the MRF1’ gene (coding for mitochondrial enoyl-ACP reductase) can be complemented by a chimeric construct coding for mitochondrially localized transgenic bacterial fabI enoyl-ACP-dehydratase- (we’ll see more about this later)

- We decided to use a chimeric construct consisting of a yeast mitochondrial localization sequence and a bacterial dehydratase (fabA) to set up a colony-color-sectoring based screen to identify the yeast mitochondrial hydroxyacyl-ACP dehydratase

Chimera

The first mention we have of the Chimera is in Book VI of the Iliad. There Homer writes that it came of divine stock and was a lion in its foreparts, a goat in the middle, and a serpent in its hindparts, and that from its mouth it vomited flames, and finally was killed by the handsome Bellerophon, the son of Glaucus, following the signs of the gods. A lion's head, goat's belly, and serpent's tail is the most obvious image conveyed by Homer's words, but Hersiod's Theogony describes the Chimera as having three heads, and this is the way it is depicted in the famous Arezzo bronze that dates from the fifth century. Springing from the middle of the animal's back is the head of a goat, while at one end it has a snake's head and at the other a lion's.

A chimeric construct produces a fusion -(usually) protein that is made up from parts of two or more different proteins. The expression has its roots in greek mythology

Construction of chimera consisting of COQ3 –mitochondrial targeting sequence fused to bacterial hydroxyacyl-ACP dehydratase expressed

from CAT1 (catalase) promoter

COQ3 mt

ADE3

2

bla pTSV30mtFabA

Strain W1536 5B/8B

ade2, ade3, leu2, trp1, ura3,

dehydratase

transform with plasmid containing mtFABA, the ADE3 gene and a selectable marker (LEU2 gene),

select on plates lacking leucine

white colonies

red colonies

dehydratase

ADE3

LEU

2

mtFABA

dehydratase

dehydratase

ADE3

LEU

2

mtFABA

Grow transformed strain on nonselective media:

plasmid is lost at a certain frequency, because the strain is viable without the plasmid.

Result: sectored colonies

sectored colonie

dehydratase

ADE3

LEU

2

mtFABA

Wild Type W1536 8B sectoring on SC- glycerol (+ 0.05% Dextrose, low Adenine)

dehydratase

ADE3

LEU

2

mtFABA

Mutagenesis (EMS): screen for mutations in the dehydratase gene that will inactivate its function

dehydratse

ADE3

LEU

2

mtFABA

Cells that lose the plasmid with mtFABA cannot survive on media containing glycerol as the sole carbon source due to lack of dehydratase activity and the resulting respiratory deficient phenotype

Cells that maintain the plasmid with mtFABA survive, because the bacterial enzyme will provide the dehydratse function

red colonies

dehydratseADE3

LEU

2

mtFABA

dehydratse

grow on media containing 3% glycerol (and 0,05% dextrose) as carbon source

Mutant 8BA#2 on SC-Glycerol (0.05% dextrose, low adenine)

Test for specificity (1) (respiratory defect): grow 2% glucose plates

ADE3 LEU

2

mtFABA

dehydratse

because glucose is a fermentable carbon source, the cells will be able to lose the mtFABA plasmid

Sectored colony

mutants that cannot lose the mtFABA plasmid are not desireable, because the lesion is likely to be in an unrelated pathway (e.g. leucine uptake)

dehydratse

white cells from colony should be respiratory deficient

Mutant 8BA#2 sectoring on SC-dextrose media (test for specificity of

mutation/mitochondrial dysfunction)

Characterization of mutants

• Test for dominance/recessiveness (mating to wild type is diploid respiratory competent or respiratory deficient)

• Complementation analysis – mating of the mutants to each other (do the mutants complement the respiratory deficient phenotype of each other are in different genes; or do the mutants not complement each other are in the same gene?)

Cloning of the mutation: transform cells with yeast genomic library and select transformants that are able lose the ADE3/mtFabA plasmid

ADE3

LEU

2

mtFABA

dehydratse

transform with genomic library, grow on 3%glycerol, 0.05% Dextrose medium (screen 20,000- 40,000 colonies)

dehydratse

URA3

Dehyratase

ADE3

LEU

2

mtFABA

cells can lose the ADE3/mtfabA plasmid if they pick up a library plasmid with an insert of a genomic fragment carrying the dehydratase gene

dehydratse ADE3

LEU

2

mtFABA

dehydratse

URA3

Dehyratase

sectored colony

isolate library plasmid, retransform to confirm ability to cause sectoring, sequence insert identify GENE! (test for activity, knockout phenotype, localization etc.)

ResultsAlexander J. Kastaniotis , Kaija J. Autio, Raija T. Sormunen and J. Kalervo Hiltunen (2004). Htd2p/Yhr067p is a yeast 3-hydroxyacyl-ACP dehydratase essential for mitochondrial function and morphology. Molecular Microbiology 53 (5), pp.1407-1421

- identified four mutants (3 a, 1α)

- all recessive

- one complementation group

- all complemented by mtFabZ (bacterial hydroxyacyl-ACP dehydratase lacking the isomerase function of FabA)

- Identified complementing genomic DNA fragment from multicopy library independently in two different mutants (three times)

Clone IV-300/10 (chromosome VIII, 230850 – 235982)

Deletions:

YHR067w DYS1

RRP4

TRM5

EcoR1/HindIIIEcoR1 Kpn1 HindIII

Xho1, Kpn1

DEco R1

DKpn1

DHindIII

Only the Kpn1 deletion construct

complements the respiratory deficient phenotype of 8BA#2

Clone IV-300/10Clone IV-300/10

YEp195 YEp195

DKpn1DKpn1DHindIII

DEcoRI

DHindIII

DEcoRI

A. SC-Glycerol B. SC-Dextrose

YHR067w is the complementing ORF!

Yhr067p has weak similarity to dehydratases

Query: 171 TVTDMDIVAYGQMSLNPHRIHWDKEYSRYVEGYDDIIMQGPFSVQLLQKCI--QPFLEQP 228 Sbjct: 17 TVTPADIALFALVSGDHNPIHVDPEFAKLA-GFPGPIAHGMLTLAIVRGLVEEQGGDNVV 75

Query: 229 IRQLRY--RNLNYIYPNTTL 246 Sbjct: 76 ARYGGWSVRFTGPVFPGDTL 95

E – value of 0.006

NCBI Conserved Domain Search

Mitochondrial extracts of cells overexpressing YHR067w exhibited increased hydratase 2 activity!

• Deletion of yhr067w reported to cause respiratory deficiency in S.cerevisiae• Predicted to be localized to the mitochondria with a probability of 0.67• The respiratory deficient phenotype of BY4741 yhr067wD is complemented by

mitochondrially localized mtFabA and mFabZ and suppressed by FAM1-1

YEpCTA1YEpCTA1

YEpFabA cyt

YEpFabA cyt

YEpFAM1-1 YEpFAM1-1

YEpmtFabZ YEpmtFabZYEpmtFabA YEpmtFabA

Clone IV-300/10

Clone IV-300/10

A. SC-Glycerol B. SC-Dextrose

A Yhr067-proA fusion localizes to mitochondria

Fig. 6. Mitochondrial localization of Yhr067-proA. FITC green fluorescent stain in BJ1991 cells expressing the Yhr067-proA fusion protein (upper left) correlates well with the Mitotracker Red stain in the same cells (upper right). Cells only expressing protein A show only a diffuse cytosolic FITC stain (lower left) that does not display any fluorescence corresponding to the Mitotracker Red stain (lower right). Exposure times were 5 s (FITC) and 150 ms (Mitotracker).

Yhr067p is the yeast mitochondrial FASII

dehydratase

The mitochondrial FAS pathway is conserved in eukaryotes

Component Yeast HumanAcetyl-CoA Carboxylase

HFA1 ACC1/2?

Phosphopantheteinyl – ACP transferase

PPT2 AASDHPPT?

Acyl Carrier Protein ACP1 NDUFAB1 Malonyl-CoA::ACP transferase

MCT1 MCAT

3-ketoacyl-ACP synthase

CEM1 OXSM

3-oxoacyl-ACP reductase

OAR1 KAR1

3-hydroxyacyl-ACP dehydratase

HTD2 HsHTD2*

2-enoyl-ACP reductase

ETR1 MECR

• After cloning of the yeast mitochondrial 3-hydroxyacyl-ACP dehydratase HTD2 gene (YHR067w):–NO OBVIOUS HOMOLOGUE IN

HUMAN GENOME!

Kaija J. Autio*, Alexander J. Kastaniotis*, Helmut Pospiech, Ilkka J. Miinalainen, Melissa S. Schonauer , Carol L. Dieckmann and J. Kalervo Hiltunen (2008). An ancient genetic link between vertebrate mitochondrial fatty acid synthesis and RNA processing. The FASEB Journal. 2008;22:569-578.)

Cloning of the human mitochondrial HTD2 analogue

• Bioinformatics no help• Cloning via functional complementation in yeast• Transformation of htd2-1 mutant with human cDNA

library in yeast expression vector; selection on plates containing glycerol as sole carbon source

• Isolation of several colonies containing complementing plasmids

• cDNA insert contained the gene encoding the RPP14 subunit of human RNase P

A second ORF on the RPP14 cDNA encoded a protein with similarity to

dehydratases

The 3’ ORF complemented the respiratory deficient phenotype

Recombinant HsHTD2 protein has hydratase-2 activity

• MBP-HsHTD2 fusion protein purified from E. coli lysate with amylose resin and followed by gel filtration

• The recombinant protein exhibited (3R)-specific hydratase 2 activity with trans-2-hexenoyl-CoA and trans-2-decenoyl-CoA substrates

Mutation of amino acids in the hydratase-2 fingerprint inactivates HsHTD2

- HsHTD2 Asp62 and His67 correspond to Asp808 and His813 of the hydratase-2

fingerprint of the Candida tropicalis MFE2 hydratase 2 domain

- Mutation of Asp62 and His67 in HsHTD2 results in inactive enzyme, as indicated by the inability to complement the yeast htd2Δ strain

HsHTD2-GFP localizes to mitochondria in HeLa cells

Expression of RPP14 and HsHTD2 in different tissues

Lei Zhang, Anil K. Joshi, Jörg Hofmann, Eckhart Schweizer, and Stuart Smith

J. Biol. Chem., Vol. 280, Issue 13, 12422-12429, April 1, 2005

Expression of human condensing

enzyme gene OXSM

Phylogenetic relationship of MaoC

dehydratases/hydratases

- Discrete clades for hydratases of multifunctional enzymes type 2 and dehydratases of eukaryotic fatty acid synthases 1

- Human mitochondrial dehydratases and other homologous eukaryotic enzymes form a branch with bacterial PhaJ hydratases - fungal mitochondrial dehydratases form a clade that is separate from the other eukaryotic mitochondrial dehydratases

The bicistronic cDNA and exon/ intron structure of RPP14/HsHTD2 are

conserved in Vertebrates

A 400 million year marriage!

• Is HsHTD2 a real protein or just pseudogene important for regulation of RPP14– A functional HTD2 open reading frame encoding

proteins localized to mitochondria has been maintained over 400 million years likely to be a protein

– Trypanosoma brucei (eukaryotic parasite) HTD2 is a close homologue to human HTD2; also other eukaryotic homologues: K. Autio, J. Guler, A. Kastaniotis, P. Englund, J. Hiltunen (2008). The 3-hydroxyacyl-ACP dehydratase of mitochondrial fatty acid synthesis in Trypanosoma brucei. FEBS Letters,582 (5): 729-733

• Is there a physiological significance to the link between these genes?

Identification of human mitochondrial ketoacyl reductase

(Chen et al., FASEB J 2009)

• MANY homologues of yeast Oar1p or E. coli fabG in mammals

• Several with clear mitochondrial import sequence

• None complementing the yeast oar1 knockout mutant

• ???!?!?• Hypothesis: Mammalian mitochondrial import

sequence is not recognized by the yeast mitochondrial protein import machinery

• Generation of chimeric proteins containing yeast mitochondrial import sequence– Two candidates complement weakly

• Hypothesis: maybe two different proteins needed in mammals for mitochondrial ketoreductase functionCoexpression of both proteins in yeastLeads to wild type complementation!

Why are two proteins needed for wild type level complementation?

• Hypothesis 1: The two proteins have different fatty acid chain length specificities; one is needed to make octanoic acid for lipoic acid synthesis, the other is required for longer fatty acids;

• Hypothesis 2: The proteins form a complexLipoic acid assay to test the hypotheses

Coexpression of the two candidates in E. coli results in the formation of an active heterotetrameric enzyme

Figure 4. Copurification of Hs17β-HSD8 and His6-HsCBR4 and the purified KARs from different organisms.

A) SDS-PAGE. Copurification of Hs17β-HSD8 and His6-HsCBR4 using Ni-NTA chromatography. Lane 1, supernatant of bacterial extract; lane 2, cell pellets; lane 3, flow through; lanes 4–9, representative fractions taken from the Ni-NTA-bound peak.

B) Copurification of Hs17β-HSD8 and His6-HsCBR4 using gel filtration chromatography. Chromatogram of the Superdex 200 size-exclusion column is shown. Insert: SDS-PAGE of fraction samples from chromatography. Lane LS: pooled samples eluted from a Ni-NTA column; lanes 15, 23, 30, 32, 34, 36, 38, 40: representative samples taken from indicated fractions.

C) Purified KARs from different organisms. Lane 1, FabG; lane 2, ScOar1p; lane 3, HsKAR (both Hs17β-HSD8 and HsCBR4 have a His6 tag); lane 4, HsKAR (only Hs17β-HSD8 His6 tagged); lane 5, HsKAR (only HsCBR4 His6 tagged).

Using mitochondrial FAS mutants to identify and characterize Mycobacterium tuberculosis

FAS II enzymes• 1: • Heterologous expression of mycobacterial proteins in yeast reveals two

physiologically functional 3-hydroxyacyl-thioester dehydratases HtdX and HtdY that are additional to HadABC and HtdZ.

• Gurvitz A, Hiltunen JK, Kastaniotis AJ.• J Bacteriol. 2009 Jan 9. [Epub ahead of print]• 2: • Function of heterologous Mycobacterium tuberculosis InhA, a type 2 fatty

acid synthase enzyme involved in extending C20 fatty acids to C60-to-C90 mycolic acids, during de novo lipoic acid synthesis in Saccharomyces cerevisiae.

• Gurvitz A, Hiltunen JK, Kastaniotis AJ.• Appl Environ Microbiol. 2008 Aug;74(16):5078-85. • 3: • Identification of a novel mycobacterial 3-hydroxyacyl-thioester

dehydratase, HtdZ (Rv0130), by functional complementation in yeast.• Gurvitz A, Hiltunen JK, Kastaniotis AJ.• J Bacteriol. 2008 Jun;190(11):4088-90.