Synthetic Genetic Array Sga Screen Biology Essay

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Genetic analysis has been used to assess gene function in vivo, identifying new components of specific pathways and ordering gene products within a pathway. Synthetic genetic interactions occur when a second mutant gene epistatically suppresses or enhances the phenotype of a particular mutant gene. A synthetic lethality genetic interaction or negative genetic interaction is observed when the combination of two otherwise viable (knockout) mutations results in a lethal phenotype (Figure 7.1) (Hartman IV et al., 2001). A less extreme form of negative genetic interaction than synthetic lethality is "synthetic sick" where genetic interaction between two knockout mutations causes an epistatic reduction in growth (Figure 7.1).

Figure 7.1: SGA plate showing Synthetic Lethal (absence of growth) and Synthetic Sick (reduced growth) Interactions

When two genes show a synthetic lethal interaction, it suggests that the gene products impact on the same vital function, such that one pathway functionally compensates for the defects in the other. In S. cerevisiae, a complete set of gene deletion mutants has been constructed for each of the about 6300 predicted genes in the genome (Winzeler et al., 1999). About 1000 genes are "essential" in that their deletion results in inviability and about 5000 genes are termed "non essential" as deletion does not result in inviability (Giaever et al., 2002, Winzeler et al., 1999). A general explanation of this remarkable result is that around 80% of the predicted genes of yeast are not required for survival being buffered by others in this function. The set of about 5000 viable deletion mutants is thus a valuable resource for systematic genetic analysis because the nature of the buffering pathways may reveal additional gene function of unnatotated or incompletely annotated ORFs (Hartman et al., 2001) (Tong et al., 2004).

Genetic interactions can be of two types (Boone et al., 2007, Lehner et al., 2006). Within-pathway (series) interactions arise between upstream or downstream components in the same pathway, or between components within the same complex (Figure 7.2). Null mutations of these generally results in phenotype suppression, as knocking out one component will render the pathway non-functional and mutations in other components will not lead to a further phenotype defect. Between-pathway (parallel) interactions on the other hand arise between components of redundant pathways that is buffering or compensating pathways. Utilising modern technologies, genome-wide screens for synthetic lethal interactions in double mutants may be performed to reveal large number of interacting pathways (Tong et al., 2001, Tong et al., 2004). The interactions of a single gene with other genes in the genome can be visualised on a genetic interaction network map, which describes interacting genes both by name and by Gene Ontology functional attributes (Ashburner et al., 2000). It has been discovered that although genes within the same pathway or complex don't undergo SL interactions, they tend to exhibit similar interaction profiles, i.e. interact with similar types of genes; a feature known as congruence (Tong et al., 2004). Furthermore, comparison of interaction networks, for example between a gene of unknown function to a gene of known function, can reveal functionally related components, as these tend to interact either directly or through bridging interactions.

Figure 7.2: Intra pathway vs. inter-pathway genetic interactions (Boone et al., 2007).

The chapter focuses on an array-based genome wide synthetic lethal analysis approach, termed synthetic genetic array (SGA) analysis (Tong et al., 2001, Tong et al., 2004), an automated method for constructing double mutants in S. cerevisiae. The availability of a haploid life cycle in yeast makes it appropriate for genetic analysis such as screens to identify synthetic lethal interactions. SGA analysis offers a well organised approach for the systematic construction of double mutants and enables a global analysis of synthetic lethal genetic interactions. The SGA screen involves crossing a single gene knockout of interest, called the "query gene" to an ordered array of about 5000 viable gene deletion mutants, and, through a series of replica-pinning steps, meiotic progeny harbouring both mutations can be identified and scored for fitness defects. It relies on the ability of yeast to grow as haploids, mate to form diploids, undergo meiosis and select for haploids that possess simultaneous disruptions of two known genes. The existence of synthetic lethal interactions between the query strain and any other non-essential gene will result in inviable meiotic progeny. Similarly, the existence of synthetic sick interactions will result in meiotic progeny with severely impaired growth. The key point of difference between SGA analysis and more traditional methods of double knock-out generation is that SGA is performed on a large-scale. This chapter utilises SGA analysis to map the genetic interactions of many genes related to ALG6 gene by genetic interaction function.

7.2 Results

7.2.1 Δalg6 SGA

The Δalg6 query strain was constructed (Materials and Methods sections 2.22.1 and 2.22.2) and mass mated with the deletion mutant array of non essential genes creating a genome-wide SGA and imaged by the laboratory digital camera imaging system (Materials and Methods section 2.23.1). A complete set of images acquired from the final SGA plates have been presented in appendices 11.6. 25 negative genetic interactions, termed hits were distinguished by SESA (Materials and Methods section 2.23.3) and are shown in Table 7.1. The 25 hits were consistent and appeared in 3 independent SGA screens performed.

Figure 7.3: Cytoscape network graph showing negative genetic interaction with ALG6. The genes are grouped based on their GO terms (cellular process) generated with BiNGO.

Table 7.1: Δalg6 gene negative interaction. GO terms and annotations are referred from the Saccharomyces Genome Database (SGD, 2009).

Gene

ORF

Name Description

Description

PMT2

YAL023C

Protein O-MannosylTransferase

Protein O-mannosyltransferase, transfers mannose residues from dolichyl phosphate-D-mannose to protein Ser/Thr residues and acts in a complex with Pmt1p

PMT1

YDL095W

Protein O-MannosylTransferase

Protein O-mannosyltransferase, transfers mannose residues from dolichyl phosphate-D-mannose to protein Ser/Thr residues

APM3

YBR288C

clathrin Adaptor Protein complex Medium chain

Mu3-like subunit of the clathrin associated protein complex (AP-3) functions in transport of alkaline phosphatase to the vacuole via the alternate pathway

NBP2

YDR162C

Nap1 Binding Protein

Protein involved in the HOG (high osmolarity glycerol) pathway, negatively regulates Hog1p by recruitment of phosphatase Ptc1p the Pbs2p-Hog1p complex

OST5

YGL226C-A

Oligosaccharyltransferase

Zeta subunit of the oligosaccharyltransferase complex of the ER lumen, which catalyzes asparagine-linked glycosylation of newly synthesized proteins

IRE1

YHR079C

Inositol REquiring

Serine-threonine kinase and endoribonuclease; transmembrane protein that mediates the unfolded protein response (UPR) by regulating Hac1p synthesis through HAC1 mRNA splicing

GOS1

YHL031C

GOlgi Snare

v-SNARE protein involved in Golgi transport, homolog of the mammalian protein GOS-28/GS28

SLT2

YHR030C

Suppression at Low Temperature

Serine/threonine MAP kinase involved in regulating the maintenance of cell wall integrity and progression through the cell cycle

BCK1

YJL095W

Bypass of C Kinase

Mitogen-activated protein (MAP) kinase acting in the protein kinase C signalling pathway

HOC1

YJR075W

Homologous to OCh1

Alpha-1,6-mannosyltransferase involved in cell wall mannan biosynthesis; subunit of a Golgi-localized complex

OPI3

YJR073C

Over Producer of Inositol

Phospholipid methyltransferase (methylene-fatty-acyl-phospholipid synthase), catalyzes the last two steps in phosphatidylcholine biosynthesis

OST3

YOR085W

OligoSaccharylTransferase

Gamma subunit of the oligosaccharyltransferase complex of the ER lumen, which catalyzes asparagine-linked glycosylation of newly synthesized proteins

BUB3

YOR026W

Budding Uninhibited by Benzimidazole

Kinetochore checkpoint WD40 repeat protein that localizes to kinetochores during prophase and metaphase

GYP1

YOR070C

Gtpase-activating protein for YPt1p

Cis-golgi GTPase-activating protein (GAP) for the Rab family members Ypt1p

CSG2

YBR036C

Calcium Sensitive Growth

Endoplasmic reticulum membrane protein, required for mannosylation of inositolphosphorylceramide and for growth at high calcium concentrations

RVS161

YCR009C

Reduced Viability on Starvation

Amphiphysin-like lipid raft protein; interacts with Rvs167p and regulates polarization of the actin cytoskeleton, endocytosis, cell polarity, cell fusion and viability following starvation or osmotic stress

OPI6

YDL096C

OverProducer of Inositol

Dubious open reading frame unlikely to encode a protein, based on available experimental and comparative sequence data; partially overlaps verified gene PMT1/YDL095W

ARO1

YDR127W

AROmatic amino acid requiring

Pentafunctional arom protein, catalyzes steps 2 through 6 in the biosynthesis of chorismate, which is a precursor to aromatic amino acids

YFL032W

YFL032W

Dubious open reading frame unlikely to encode a protein, based on available experimental and comparative sequence data; partially overlaps the verified gene HAC1/YFL031W

PML39

YML107C

Pre-mRNA Leakage

Protein required for nuclear retention of unspliced pre-mRNAs along with Mlp1p and Pml1p; anchored to nuclear pore complex via Mlp1p and Mlp2p

LSM7

YNL147W

Like SM

Lsm (Like Sm) protein; part of heteroheptameric complexes

YNL296W

YNL296W

Dubious open reading frame unlikely to encode a functional protein; deletion adversely affects sporulation; deletion mutant exhibits synthetic phenotype under expression of mutant huntingtin fragment

YGL024W

YGL024W

Dubious open reading frame unlikely to encode a protein, based on available experimental and comparative sequence data; partially/completely overlaps the verified ORF PGD1/YGL025C

ROM2

YLR371W

RhO1 Multicopy suppressor

GDP/GTP exchange protein (GEP) for Rho1p and Rho2p

URE2

YNL229C

UREidosuccinate transport

Nitrogen catabolite repression transcriptional regulator that acts by inhibition of GLN3 transcription in good nitrogen source

The Δalg6 SGA resulted in 25 negative interactions. When analysed with SGD gene ontology OST3, OST5, HOC1, PMT1, PMT2, BCK1, IRE1, ARO1, OPI3, SLT2 genes were involved in transferase activity.

OST5 and OST3 contribute to dolichyl-diphosphooligosaccharide-protein glycotransferase activity. OST5 is the zeta subunit of the oligosaccharyltransferase complex of the ER lumen, which catalyzes asparagine-linked glycosylation of newly synthesized proteins. OST3 is the Gamma subunit of the oligosaccharyltransferase complex of the ER lumen, which catalyzes asparagine-linked glycosylation of newly synthesized proteins; Ost3p and Ost5p proteins are important for N-glycosylation.

HOC1 catalyses the transfer of a mannose residue from GDP-mannose to an oligosaccharide, forming an alpha-1,6-linkage. Alpha-1,6-mannosyltransferase is involved in cell wall mannan biosynthesis; subunit of a Golgi-localized apparatus that also contains Anp1, Mnn9, Mnn11, and Mnn10 proteins.

PMT1 and PMT2 contribute to dolichyl-phosphate-mannose-protein mannosyltransferase activity. They catalyse the reaction: dolichyl phosphate D-mannose + protein = dolichyl phosphate + O-D-mannosylprotein. PMT1 is involved in Protein O-mannosyltransferase and transfers mannose from dolichyl phosphate-D-mannose to protein Ser/Thr residues and is essential for cell wall rigidity. Pmt1p is involved in ER quality control (Strahl-Bolsinger S, et al., 1993) (Goder V et al., 2011). PMT2 is also involved in protein O-mannosyltransferase transfers mannose residues from dolichyl phosphate-D-mannose to protein Ser/Thr residues. Pmt2p is also involved in ER quality control and acts in a complex with Pmt1p (Goder V et al., 2011).

BCK1 Catalyses the phosphorylation of an amino acid residue in a protein, usually according to the reaction: a protein + ATP = a phosphoprotein + ADP. Upon activation by Bkc1p phosphorylates downstream kinases Mkk1p and Mkk2p (Heinisch JJ, et al., 1999).

IRE1 is a Serine-threonine kinase and endoribonuclease transmembrane protein that mediates the unfolded protein response (UPR) by regulating Hac1p synthesis through HAC1 mRNA splicing. Kar2p binds inactive Ire1p and releasing it upon ER stress (Sidrauski C, et al., 1997) (Welihinda AA, et al., 1996).

ARO1 catalyzes in the biosynthesis of chorismate, which is a precursor to aromatic amino acids. OPI3 is responsible for Phospholipid methyltransferase (methylene-fatty-acyl-phospholipid synthase), catalyzes the last two steps in phosphatidylcholine biosynthesis (Duncan K, et al., 1987) (Jones EW, et al., 1982).

GOS1, IRE1, RVS161 and URE2 were involved in specific protein binding activity. GOS1 interacts selectively with one or more SNAREs on another membrane to mediate membrane fusion. GOS1 is a v-SNARE protein involved in Golgi transport, homolog of the mammalian protein GOS-28/GS28. IRE1 interacts selectively and non-covalently with unfolded proteins. RVS161 interacts selectively and non-covalently with any protein component of cytoskeleton (actin, microtubule, or intermediate filament cytoskeleton). Rvs161p is a amphiphysin-like lipid raft protein. It interacts with Rvs167p and regulates polarization of the actin cytoskeleton, endocytosis, cell polarity, cell fusion and viability following starvation or osmotic stress. URE2 interacts selectively and non-covalently with a phosphorylated protein.

CSG2 encodes an endoplasmic reticulum membrane protein, required for mannosylation of inositolphosphorylceramide and for growth at high calcium concentrations. GYP1 increases the rate of GTP hydrolysis by a GTPase of the Rab family. It is a Cis-golgi GTPase-activating protein (GAP) for the Rab family members Ypt1p and is involved in vesicle docking and fusion. ROM2 stimulates the exchange of guanyl nucleotides associated with a GTPase. Under normal cellular physiological conditions, the concentration of GTP is higher than that of GDP, favouring the replacement of GDP by GTP in association with the GTPase.

7.2.1.1 Linkage group genes surrounding ALG6 gene

A standard feature of SGAs is a "linkage group" of negative genetic interactions of genes surrounding the query gene which arises by linkage disequilibrium of genes that are immediately physically and numerically contiguous to the query gene. Since recombination within short distances in a genome is unlikely, these genes take on the "hit" characteristics of the query gene in its genetic interactions. On face value, the linkage group is an SGA artefact. However, it is a useful artefact for verification of the proper insertion of the query gene knockout at its correct genomic position and is therefore described below.

Figure 7.4: Δalg6 SGA showing linkage group genes. From top left: YOR006C, YOR005C, YOR003W, YOR002W (ALG6), YOR001W, YOR016C, YOR015W, YOR014W, YOR013W, YOR012W, YOR011W, YOR010C, YOR009W, YOR008C-A, YOR007C, YNR050C, YOR027W, YOR026W, YOR025W, YOR024W, YOR023C, YOR022C, YOR021C, YOR019W, YOR018W, YOR017W, YOR035C, YOR034C, YOR033C, YOR032C, YOR031W, YOR030W, YOR029W, YOL002C, YOL001W, YOL013W-A, YOL013C, YOL012C, YOL011W, YOL009C, YOL008W, YOL007C, YOL006C, YOL004W, YOL003C. Δalg6 gene shown in yellow box and linkage group gene shown in red boxes. The discontinuous linkage disequilibrium was a result of the yeast DMA not arrayed exactly contiguously by adjacent gene number for this section of the array.

As described above, SGAs typically displays query gene hits with a chromosomally contiguous set of genes that are physically adjacent to the query gene locus (YOR002W) that do not recombine. Genes of this linkage group are not counted as SGA epistatic hits. In the case of ALG6 the interesting long linkage group observed in Figure 7.4 was a result of ALG6 gene being adjacent to the centromere of yeast chromosome XV where recombination is even less frequent than the genome as a whole.

Figure 7.5: Yeast Chromosome XV features that span coordinates 319417 - 341051 bp. The Black dot represents the centromere (www.yeastgenome.org)

This linkage group for Δalg6 query strain appeared in all three SGA screen repeats, thus confirming that ALG6 gene was replaced with the NatMX4 cassette (Δalg6::NatR) (Figure 7.5).

7.2.2 CDG-Ic SGA

The humanised CDG-Ic query strain was constructed (Materials and Methods sections 2.22.3 and 2.22.4) and mass mated with the deletion mutant array of non essential genes creating a genome-wide SGA (Materials and Methods section 2.23.1). 25 negative interactions were scored (hits) and are shown in Table 7.2. These hits appeared in 3 independent SGA screens performed. A complete set of images acquired from the final SGA plate have been presented in appendices 11.7.

Figure 7.6: Cytoscape network graph showing negative genetic interaction with CDG-Ic Disorder. The genes are grouped based on their GO terms (cellular process) generated with BiNGO.

Table 7.2: Human ALG6 gene (C998T point mutation) negative interaction. GO terms and annotations are referred from the Saccharomyces Genome Database (SGD, 2009).

Gene

ORF

Name Description

Description

PMT2

YAL023C

Protein O-MannosylTransferase

Protein O-mannosyltransferase, transfers mannose residues from dolichyl phosphate-D-mannose to protein Ser/Thr residues and acts in a complex with Pmt1p

PMT1

YDL095W

Protein O-MannosylTransferase

Protein O-mannosyltransferase, transfers mannose residues from dolichyl phosphate-D-mannose to protein Ser/Thr residues

APM3

YBR288C

clathrin Adaptor Protein complex Medium chain

Mu3-like subunit of the clathrin associated protein complex (AP-3); functions in transport of alkaline phosphatase to the vacuole via the alternate pathway

NBP2

YDR162C

Nap1 Binding Protein

Protein involved in the HOG (high osmolarity glycerol) pathway, negatively regulates Hog1p by recruitment of phosphatase Ptc1p the Pbs2p-Hog1p complex

RAD4

YER162C

RADiation sensitive

Protein that recognizes and binds damaged DNA (with Rad23p) during nucleotide excision repair; subunit of Nuclear Excision Repair Factor 2 (NEF2); homolog of human XPC protein

OST5

YGL226C-A

OligoSaccharylTransferase

Zeta subunit of the oligosaccharyltransferase complex of the ER lumen, which catalyzes asparagine-linked glycosylation of newly synthesized proteins

IRE1

YHR079C

Inositol REquiring

Serine-threonine kinase and endoribonuclease; transmembrane protein that mediates the unfolded protein response (UPR) by regulating Hac1p synthesis through HAC1 mRNA splicing

SLT2

YHR030C

Suppression at Low Temperature

Serine/threonine MAP kinase involved in regulating the maintenance of cell wall integrity and progression through the cell cycle

BCK1

YJL095W

Bypass of C Kinase

Mitogen-activated protein (MAP) kinase acting in the protein kinase C signalling pathway

HOC1

YJR075W

Homologous to OCh1

Alpha-1,6-mannosyltransferase involved in cell wall mannan biosynthesis; subunit of a Golgi-localized complex

OPI3

YJR073C

OverProducer of Inositol

Phospholipid methyltransferase (methylene-fatty-acyl-phospholipid synthase), catalyzes the last two steps in phosphatidylcholine biosynthesis

OST3

YOR085W

OligoSaccharylTransferase

Gamma subunit of the oligosaccharyltransferase complex of the ER lumen, which catalyzes asparagine-linked glycosylation of newly synthesized proteins

BUB3

YOR026W

Budding Uninhibited by Benzimidazole

Kinetochore checkpoint WD40 repeat protein that localizes to kinetochores during prophase and metaphase

GYP1

YOR070C

Gtpase-activating protein for YPt1p

Cis-golgi GTPase-activating protein (GAP) for the Rab family members Ypt1p

CSG2

YBR036C

Calcium Sensitive Growth

Endoplasmic reticulum membrane protein, required for mannosylation of inositolphosphorylceramide and for growth at high calcium concentrations

RVS161

YCR009C

Reduced Viability on Starvation

Amphiphysin-like lipid raft protein; interacts with Rvs167p and regulates polarization of the actin cytoskeleton, endocytosis, cell polarity, cell fusion and viability following starvation or osmotic stress

OPI6

YDL096C

OverProducer of Inositol

Dubious open reading frame unlikely to encode a protein, based on available experimental and comparative sequence data; partially overlaps verified gene PMT1/YDL095W

ARO1

YDR127W

AROmatic amino acid requiring

Pentafunctional arom protein, catalyzes steps 2 through 6 in the biosynthesis of chorismate, which is a precursor to aromatic amino acids

YFL032W

YFL032W

Dubious open reading frame unlikely to encode a protein, based on available experimental and comparative sequence data; partially overlaps the verified gene HAC1/YFL031W

PML39

YML107C

Pre-mRNA Leakage

Protein required for nuclear retention of unspliced pre-mRNAs along with Mlp1p and Pml1p; anchored to nuclear pore complex via Mlp1p and Mlp2p

LSM7

YNL147W

Like SM

Lsm (Like Sm) protein; part of heteroheptameric complexes

YNL296W

YNL296W

-

Dubious open reading frame unlikely to encode a functional protein; deletion adversely affects sporulation; deletion mutant exhibits synthetic phenotype under expression of mutant huntingtin fragment

YGL024W

YGL024W

-

Dubious open reading frame unlikely to encode a protein, based on available experimental and comparative sequence data; partially/completely overlaps the verified ORF PGD1/YGL025C

ROM2

YLR371W

RhO1 Multicopy suppressor

GDP/GTP exchange protein (GEP) for Rho1p and Rho2p

URE2

YNL229C

UREidosuccinate transport

Nitrogen catabolite repression transcriptional regulator that acts by inhibition of GLN3 transcription in good nitrogen source

The CDG-Ic SGA resulted in 25 negative interactions. GOS1 did not show negative interaction in the CDG-Ic yeast strain as seen with Δalg6 yeast strain. On the other hand RAD4 was the only additional negative interaction. Rad4p recognizes and binds damaged DNA (with Rad23p) during nucleotide excision repair. It is a subunit of Nuclear Excision Repair Factor 2 (NEF2), a homolog of human XPC protein.

The genes that appeared as hits in both Δalg6 and CDG-Ic SGA are depicted in a network graph shown in Figure 7.3 and 7.6, generated by Cytoscape (Cline et al., 2007). The network diagram shows that except for one gene, the negative interactions are identical between Δalg6 and CDG-Ic SGA. The similarity between the two SGA suggests the adverse effect of the A333V substitution onto human Alg6p function. The proximity of the A333V mutation to a large domain of the Alg6 protein that is conserved between yeast and man suggests a possible alteration of the catalytic properties of Alg6p.

7.2.2.1 Linkage group genes surrounding human ALG6 gene (C998T point mutation)

The Linkage Group for C998T point mutation on the human ALG6 gene appeared in all three SGA screen repeats, thus confirming C998T point mutation on human ALG6 linked to the NatMX4 cassette (Δalg6::human ALG6(C998T)-NatR) (Figure 7.7).

Figure 7.7: CDG-Ic SGA showing linkage group genes. From top left: YOR006C, YOR005C, YOR003W, human ALG6 gene (C998T point mutation), YOR001W, YOR016C, YOR015W, YOR014W, YOR013W, YOR012W, YOR011W, YOR010C, YOR009W, YOR008C-A, YOR007C, YNR050C, YOR026W, YOR025W, YOR024W, YOR023C, YOR022C, YOR021C, YOR019W, YOR018W, YOR017W, YOR037W, YOR035C, YOR034C, YOR033C, YOR032C, YOR031W, YOR030W, YOL002C, YOL001W, YOL013W-A, YOL013C, YOL012C, YOL011W, YOL009C, YOL008W, YOL007C, YOL006C, YOL004W, YOL003C. Human ALG6 gene (C998T point mutation) shown in yellow box and linkage group gene shown in red boxes.

7.3 Comparison of negative genetic interactions between this study and the Saccharomyces Genome Database

The negative interactions obtained in this study using Δalg6 or CDG-Ic as query genes were compared to the data presented for ALG6 double mutants with non-essential genes in Saccharomyces Genome Database.

Table 7.3: Comparison of negative interactions between this study and the Saccharomyces Genome Database

Δalg6 negative interactions

CDG-Ic negative interactions

SGD total Δalg6 negative interactions

PMT2 PMT1 APM3

NBP2 OST5 IRE1

SLT2 BCK1 HOC1 OPI3 OST3 BUB3 GYP1 GOS1 CSG2

RVS161 OPI6 ARO1

YFL032W PML39

LSM7 YNL296W

YGL024W ROM2

URE2

PMT2 PMT1 APM3

NBP2 OST5 IRE1

SLT2 BCK1 HOC1 OPI3 OST3 BUB3 GYP1 RAD4 CSG2

RVS161 OPI6 ARO1

YFL032W PML39

LSM7 YNL296W

YGL024W ROM2

URE2

PMT2 PMT1 APM3 NBP2 OST5 IRE1 SLT2 BCK1 HOC1 OPI3 OST3 BUB3 GYP1 GOS1 RAD4 HAC1 GUP1 GET2 GAS1 DGK1 IRC23 OST6 PMR1 BST1 APL5 CUE5 SPF1 LAS21 RSB1 SAC1 GET1 PER1 ARL3 APL6 ALG8 KEX1 ARL1 ERG4 RHB1 TFP1 IZH1 ANP1 RTF1 KIP3 CLC1 MNN11 FAR3 SCJ1 KRE1 ISW2 ERR1 ATG11 ERV15 VMA9 ERV41 YEL057C YOR390W

This comparison does not include negative genetic interactions involving the Δalg6 and the CDG-Ic query genes and S. cerevisiae essential genes since these normally inviable strains require means of conditionally controlling the knockout phenotype. Essential gene double mutants with Δalg6 listed on SGD are: WBP1 DPM1 GPI8 OST1 PKC1 STT3 GAB1 SWP1 OST2 DED1 SEC63.

For the 25 non-essential genes, negative interactions observed as double mutants with Δalg6 SGA and CDG-Ic SGA, 15 interactions were reported previously in literature (Saccharomyces Genome Database) suggesting that the genome-wide SGA approach used in this study to characterise CDG-Ic was an effective means of exploring functions related to disease mutation e.g.(protein glycosylation, ER stress). The other 11 (44%) interactions observed in this study were found in both the SGA screens. GOS1 was observed as a hit between Δalg6 SGA and SGD. Similarly RAD4 was observed as a hit between CDG-Ic SGA and SGD. Overall, 96% of the hits obtained were identical between Δalg6 SGA and CDG-Ic SGA (Figure 7.8).

Figure 7.8: Venn diagram representing overlaps between Δalg6 and CDG-Ic negative interactions. 15 interactions represented in bold are reported in SGD. GOS1 was observed as a hit between Δalg6 SGA and SGD (blue). Similarly RAD4 was observed as a hit between CDG-Ic SGA and SGD (red).

The network of genetic interactions with alg6 and CDG-Ic are highly enriched for genes involved in processes such as glycosylation. Genes involved in transferase activity (OST3, OST5, HOC1, PMT1, PMT2, BCK1, IRE1, ARO1, OPI3 and SLT2) appeared in both the screens, suggesting an identical mechanism involved in the glycosylation steps in the endoplasmic reticulum between yeast and humans. Also the shared genes involved in stress response (BCK1, IRE1, NBP2, PMT1, PMT2, RAD4, ROM2, RVS161, SLT2) indicate involvement of stress as a result of misglycosylation either by deletion of the ALG6 gene or the adverse effect of C998T point mutation on the humanised ALG6 gene in yeast. This shows that the ALG6 genetic network was highly involved in multiple processes that can work together to alleviate unfolded protein stress within the cell, such as glycosylating proteins in attempts to achieve correct folding and eliminating terminally misfolded proteins. To observe yeast double mutants involved in different cellular pathway, genes involved in negative genetic interactions were analysed by the SGD GO mapper programme. A complete list of genes involved in glycosylation interacting in multiple patways is presented in appendices 11.8. Three important pathways showed involvement of genes obtained as negative interactions in our study namely genes involved in carbohydrate metabolism process (6 genes), protein modification process (8 genes) and response to stress (9 genes) as described in the next section.

7.4 Genes involved in GO term "carbohydrate metabolism process"

In yeast, carbohydrate metabolism processes include protein glycosylation, an enzymatic process that attaches glycans to proteins, cellular polysaccharide metabolic process involved in cell wall mannan biosynthesis and inositol metabolic process, a pathways involving inositol as shown in the following diagram.

Figure 7.9: Genes (HOC1, IRE1, OST3, OST5, PMT1, and PMT2) involved in cellular carbohydrate metabolic process. The green boxes indicate pathways involved in cellular carbohydrate metabolism and grey boxes indicate specific genes related to the respective processes.

The network of genetic interactions with alg6 and CDG-Ic are highly enriched for genes involved in Cellular Carbohydrate metabolism processes (Figure 6.9) including OST3 and OST5 involved in protein N-linked glycosylation; PMT1 and PMT2 involved in protein O-linked glycosylation; HOC1 involved in cellular polysaccharide metabolic process. Deletion of any one of the above genes with Δalg6 or CDG-Ic leads to perturbation of the carbohydrate metabolism processes synthetic lethal negative genetic interactions.

7.5 Genes involved in protein modification process

Subunits Ost3 and Ost6 are present in the yeast OST complex and they modify the transfer specificity towards proteins to be glycosylated and they specify the interaction with different translocation complexes. A family of PMT genes (protein O-mannosyl transferases) was discovered in yeast (Gentzsch M, et al., 1996). The characterization of the non-lethal pmt mutants showed O-mannosylation to play a critical role in a number of important physiological processes, such as in polar cell growth and in the formation of an intact cell wall, an essential protein modification.

Figure 7.10: Genes (BCK1, IRE1, OST3, OST5, PMT1, PMT2, SLT2, URE2) involved in protein modification process. Green boxes indicate pathways involved in cellular protein modification and grey boxes indicate specific genes elucidated here by SGA related to respective processes.

Protein phosphorylation is a post-translational modification of proteins in which a serine, threonine or tyrosine residue is phosphorylated by a protein kinase by the addition of a covalently bound phosphate group. Regulation of proteins by phosphorylation is one of the most common modes of regulation of protein function. In almost all cases of phosphoregulation, the protein switches between a phosphorylated and an unphosphorylated form, and one of these two is an active form. Bypass of C Kinase (BCK1) is a mitogen-activated protein kinase acting in the protein kinase C signalling pathway, which controls cell integrity. Upon activation by Pkc1p phosphorylates downstream kinases Mkk1p and Mkk2p. Protein urmylation is the covalent attachment of the ubiquitin-like protein URM1 to another protein. Urmylation pathway is involved in nutrient sensing. Ubiquitin is a small modifier protein that is conjugated to substrates to target them for degradation.

7.6 Genes involved in response to stress

The network of genetic interactions with alg6 and CDG-Ic are highly enriched for genes involved in response to stress (Figure 7.11). The mammalian unfolded protein response protects the cell against the stress of misfolded proteins in the endoplasmic reticulum. Upon sensing unfolded proteins, the ER transmembrane endoribonuclease and kinase, IRE1 oligomerizes and is activated by autophosphorylation and uses its endoribonuclease activity to excise an intron from the mRNA of the transcription factor Hac1p in yeast. This basic leucine zipper transcription factor regulates the unfolded protein response via UPRE binding and membrane biogenesis.

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Figure 7.11: Genes (BCK1, IRE1, NBP2, PMT1, PMT2, RAD4, ROM2, RVS161, SLT2) involved in stress response. Green boxes indicate pathways involved in response to stress and grey boxes indicate genes elucidated by the SGA reported here related to respective processes.

In addition to enrichment of genes involved in response to protein misfold, other genes involved in response to stress were observed like RVS161 and NBP2 showing enrichment to osmotic stress.

7.7 Conclusion

The aim of this chapter was to study the genetic networks surrounding the alpha 1,3 glucosyltransferase, a specific enzyme of N-linked glycosylation in cells. This chapter elucidated the involvement of numerous genes involved in cellular carbohydrate metabolic process and protein modification process. In addition genes enriched for response to stress were observed. SGA analysis between ALG6 and CDG-Ic disorder showed major overlap in their genetic interactions indicating that alg6 knockout and the A333V amino acid substitution severely impaired the ALG6 protein function.

The ALG6 deficiencies in the CDG-Ic patient could occur because secretory and membrane proteins require glycosylation for correct folding affecting function and/or transport through the secretory pathway. In mammalian cells, calnexin and calreticulin seem to be important for quality control. They are lectins that interact specifically with partially trimmed, monoglucosylated N-linked oligosaccharides. Thus, the absence of glucose on the N-linked chains can alter their association with the quality control apparatus, which can delay their movement through the secretory pathway or lead to their degradation in the endoplasmic reticulum (Clerc et al., 2009). Furthermore, the oligosaccharyl transferase complex, responsible for the transfer of the sugar chains to the polypeptide, has a decreased efficiency for transferring the incomplete oligosaccharide substrate to the protein backbone. Thus, complete glucosylation is crucial for efficient transfer of the lipid linked oligosaccharide to the protein and thereafter for proper quality control.

The results from this chapter and the previous Chapters taken together allowed arguing, it was valid to seek a pharmacological chaperone to rescue the CDG-Ic mutant disorder.

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