An intact COP9 signalosome promotes increased protein amounts of septins and

Proteins, which abundances are increased in ΔcsnE compared to ΔlysA and csnE complementation strain are listed in Table 12. Thereby, only the fold change of proteins in ΔcsnE compared to the ΔlysA background strain are shown. The fold changes are in a similar magnitude if they are compared to the csnE^comp strain. These as well as the fold changes for the control csnE^comp/ΔlysA are listed in Supplementary Table S2.

A majority of these proteins was categorized to fungal development (Figure 17). Among these 15 proteins, four septins were identified. In the absence of a functional COP9 signalosome, septin proteins are reduced in their protein abundance. Furthermore, three proteins namely ANIA_08547, ANIA_06048, ANIA_03636 and Nmt1 are involved in hyphal growth or early stages of conidiophore development, whereas ANIA_08547 has the highest log₂ fold change.

Table 12: Proteins with decreased abundance during vegetative growth in a ΔcsnE strain.

Log₂ SILAC ratios of ΔcsnE/ΔlysA are given in this table. Low values indicate downregulation of protein abundances in the deletion of csnE compared to the wild type like background strain.

The intensity of the blue color displays the level of change in protein abundance. The description of identified proteins derived from information given from AspGD and FungiDB (Cerqueira et al., 2014; Stajich et al., 2012). SILAC ratios marked with * could not be exclusively attributed to one protein. The bold line indicates the threshold between minor decreased protein abundance and a stronger decrease in protein abundance. This threshold equals the SILAC ratio described in

-2.69 ANIA_08547 Putative glucose-methanol-choline (GMC) oxidoreductase, required for early stages of conidiophore development -2.64 ANIA_03344 Putative GNAT-type acetyltransferase

-2.62 ANIA_10296 Ortholog(s) have fumarate reductase (NADH) activity -2.19 ANIA_06058 DUF833 domain-containing protein, orthologs have function

in filamentous growth

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-1.99 ANIA_03524 Ngn27, acetyltransferase, galactonate metabolism -1.58 ANIA_03674 Domains with predicted phospholipid binding activity -1.52 ANIA_05823 SidA, L-ornithine N5 monooxygenase

-1.35 ANIA_00858 Hsp104, heat shock -1.28 ANIA_09180 Putative transketolase -1.23 ANIA_07062 Uncharacterized

-1.19 ANIA_08009 Nmt1, thiamine biosynthesis process, hyphal growth

-1.18 ANIA_09148 GalF, putative UTP-glucose-1-phosphate uridylyltransferase -1.18 ANIA_08145 Uncharacterized, putative AN8142 cluster

-1.17 ANIA_04727 UgeA, UDP-glucose 4-epimerase, galactose metabolism -1.13 ANIA_03012 KapL, role in protein export from nucleus, tRNA re-export

from nucleus and cytosol, nuclear envelope localization -1.11 ANIA_04923 Putative 3-hydroxy-3-methylglutaryl coenzyme A synthase

with a predicted role in sterol metabolism -1.04 ANIA_03906 Uncharacterized

-1.04 ANIA_03223 PfkA, putative 6-phosphofructokinase with a predicted role in gluconeogenesis and glycolysis

-1.01 ANIA_04908 Putative eukaryotic translation initiation factor subunit -0.99 ANIA_10709 GfaA, putative glutamine-fructose-6-phosphate

transaminase -0.93 ANIA_08182 AspC, septin

-0.93 ANIA_01394 AspD, putative septin

-0.93 ANIA_05586 Putative mannose-1-phosphate guanylyltransferase with a predicted role in mannose, fructose,sorbitol metabolism -0.92 ANIA_06004 Protein with an RNA recognition motif, asperthecin cluster -0.92 ANIA_01158 Ortholog(s) have mRNA 3'-UTR, mRNA 5'-UTR binding,

translation repressor activity, nucleic acid binding activity -0.92 ANIA_02532 Ortholog(s) have copper ion binding, primary amine oxidase

activity

-0.88 ANIA_08815 Putative isoflavone reductase family protein -0.87 ANIA_07262 Uncharacterized

-0.86 ANIA_03112 UgmA, UDP-galactopyranose mutase, enzyme in galactofuranose biosynthesis, cell wall

-0.84 ANIA_06688 AspB, putative septin B

-0.84 ANIA_04042 Putative C-22 sterol desaturase, sterol metabolism

-0.83 ANIA_03829 Putative succinate-semialdehyde dehydrogenase [NAD(P)+]

with a predicted role in 4-aminobutyrate (GABA) shunt

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-0.83 ANIA_04591 Ortholog(s) have phosphopentomutase activity, role in guanosine catabolic process

-0.81 ANIA_05895 GdiA, putative Rab GDP-dissociation inhibitor -0.80 ANIA_04463 Uncharacterized

-0.80 ANIA_03636 Ortholog(s) have role in cellular response to biotic stimulus, cellular response to starvation and filamentous growth -0.80 ANIA_02925 PexF, putative peroxisomal import protein (peroxin) with a

role in fatty acid utilization

-0.79 ANIA_08233 Ortholog(s) have phosphatidylinositol transporter activity -0.79 ANIA_06341 Similarity to S. cerevisiae Crn1p (cytoskeletal component) -0.78 ANIA_06266 Uncharacterized

-0.77 ANIA_04667 AspA, septin

-0.76 ANIA_02756 SlaB, predicted actin binding protein

-0.75 ANIA_10614 Ortholog(s) have G-quadruplex DNA binding, ribosome binding, telomeric DNA binding, triplex DNA binding activity -0.74 ANIA_08012 Putative bifunctional enzyme with a predicted role in sterol

metabolism

-0.73 ANIA_03026 CopA, alpha-COP coatamer-related protein involved in the establishment and maintenance of polarized growth -0.71 ANIA_09094 UngA, putative UDP-N-acetylglucosamine

pyrophosphorylase with role in chitin biosynthesis

-0.70 ANIA_02867 PgmB, putative phosphoglucomutase with a predicted role in carbohydrate metabolism

-0.63 ANIA_07146 Putative S-adenosyl-methionine

delta-24-sterol-C-methyltransferase with a predicted role in sterol metabolism -0.61 ANIA_01810 OtaA, ornithine transaminase, involved in utilization of

arginine as a proline source -0.59 ANIA_00327

Ortholog(s) have ATP-dependent 3'-5' DNA helicase activity, ATP-dependent 5'-3' DNA helicase activity and role in box C/D snoRNP assembly, histone exchange, rRNA processing

-0.55 ANIA_02436 AclB, putative ATP citrate synthase with a predicted role in TCA intermediate metabolism

-0.54 ANIA_07011 Uncharacterized -0.54 ANIA_07208 SET domain protein

-0.50 ANIA_02435 AclA, putative ATP citrate synthase with a predicted role in TCA intermediate metabolism

80

Only 21 % of the proteins that are decreased in their abundance in ΔcsnE belong to primary metabolism. This encompasses the categories electron (e^-) transfer, cofactors, vitamin biosynthesis; carbohydrate metabolism and amino acid metabolism. Proteins related to transcriptional control, mRNA translation or nuclear transport are decreased in their abundance in ΔcsnE.

Figure 17: Classification of proteins, which abundances are increased by CsnE.

Proteins listed in Table 12 were assigned to different categories. The number behind the category indicates the number of proteins assigned to this class. One fourth of all proteins decreased in the abundance by the presence of CsnE were classified to fungal development.

In summary, relative quantification of protein abundances using SILAC is applicable with the filamentous fungus A. nidulans using an L-lysine auxotrophic strain. Abundances of 13.2 % of the proteins identified in this study are changed in a csnE deletion mutant during vegetative growth conditions. Thereby, CsnE decreases the abundance of approximately 5 % of these proteins, which were categorized to approximately two thirds to primary metabolism.

Furthermore, nearly 8 % of the identified proteins were increased in their abundance by CsnE.

Thereby, four septins and proteins involved in hyphal growth or cytoskeleton related proteins were identified. Through the regulation of the UPS by deneddylation of CRLs, CsnE influences the abundance of proteins already during fungal vegetative growth conditions.

Vegetative growth in the presence of a functional COP9 signalosome supports an increased steady state level of specific developmental proteins and a reduced steady state level of proteins with a role in amino acid metabolism as major effect on the fungal proteome.

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