LC-MS analysis identifies potential Spp1 interaction partners

Besides the initially described function of signal peptide peptidases (SPP) in cleaving left-over signal peptides in the ER membrane, only a little is known about other potential substrates and the contribution of their cleavage products in cellular processes (Voss et al., 2013). Since Spp1 seems not to be involved in ERAD (Chapter 3.3.7), H2O2 detoxification (Chapter 3.3.6) and growth under hypoxia (Chapter 3.3.7), a co-immunoprecipitation assay with subsequent liquid chromatography-mass

spectrometry (LC-MS) analysis was performed to identify possible interaction partners of Spp1 in U. maydis. To this end, strains were generated expressing the Spp1-GFP fusion protein and the catalytically inactive Spp1^D279A-GFP mutant in the SG200∆spp1 background. Both fusion proteins were expressed under the control of the constitutive active otef promoter. The catalytically inactive Spp1^D279A mutant was used to inhibit substrate cleavage and by this increase the probability to identify putative protease/substrate interactions. These protein interactions are transient and often hard to detect using immunoprecipitation techniques. To identify unspecific protein binding to the GFP tag, the strain SG200 Potef:GFP-3xHA served as a negative control. The strains SG200∆spp1 P^otef:spp1-GFP, SG200∆spp1 Potef:spp1^D279A-GFP and SG200 Potef:GFP-3xHA were grown in CMG to an OD600 of 0.35 and treated 4 hours with or without TM for UPR induction. Protein extracts were subjected to immunoprecipitation using magnetic agarose GFP-trap beads. For quality control, an immunoblot was performed prior to LC-MS analysis to check for sufficient protein levels in the untreated conditions, which was indeed the case (Fig 3.42).

Figure 3.42: Western hybridization of pull-down of Spp1-GFP and Spp1^D279A-GFP. Input and eluate fractions of the untreated conditions were used for quality control prior to LC-MS analysis. Samples were resolved by SDS-PAGE (10%) and analyzed by Western hybridization. For detection of the Cib1-GFP fusion protein, a GFP specific antibody was used. The asterisk indicates the Spp1-GFP fusion protein with a predicted size of ~73 kDa. Data generated in (Hach, 2018).

In the LC-MS analysis, a total number of 11 proteins were co-immunoprecipitated with Spp1-GFP and/or Spp1^D279A-GFP. The proteins UMAG_02016 (Fig 3.43, not regulated in planta), UMAG_02266 (Fig 3.43, downregulated in planta), UMAG_04994 (Fig 3.43, upregulated in planta) and UMAG_06273 (Fig 3.43, not regulated in planta) were identified in the treated and untreated conditions of Spp1-GFP and Spp1^D279A-GFP at least twice. A BLASTp analysis revealed that UMAG_02016 has a predicted Cytochrome b5-like Heme/Steroid binding domain (E-value 1.25x10^-9) and is related to Dap1p of S. cerevisiae (Similarity 66%, E-value 4x10^-31). In Schizosaccharomyces pombe, Dap1 interacts with

62 of S. cerevisiae (Similarity 47%, E-value 7x10^-26). The protein UMAG_10649 is related to an oligosaccharyltransferase complex subunit delta (ribophorin II) (Similarity 44%, E-value 1x10^-15) in Cryptococcus gattii. Members of the oligosaccharyltransferase (OST) protein complex are essential for N-linked glycosylation of proteins (Kelleher and Gilmore, 1994). The protein UMAG_06273 has a flavodoxin domain (E-value 1.37x10^-36) as well as an NADPH cytochrome p450 reductase (CYPOR) domain (E-value 0) and is related to Ncp1p of S. cerevisiae (Similarity 56%, E-value 5x10^-152), which is involved sterol biosynthesis. The protein UMAG_02833 is related to the S. cerevisiae Sec66p (Similarity 55%, E-value 2x10^-1) that is part of the Sec63 translocation complex (Feldheim et al., 1993).

UMAG_05433 has a PMT1 domain (E-value 0) and is related to the dolichyl-phosphate-mannose-protein O-mannosyl transferase Pmt4p of S. cerevisiae (Similarity 62%, E-value 0) and is required for O-linked glycosylation of secretory and cell surface proteins (Sanders et al., 1999). The protein UMAG_11590 has a PqqL domain and is predicted to be a Zn-dependent peptidase (E-value 7.34x10

-111). The protein is related to Mas1p of S. cerevisiae (Similarity 72%, E-value 9x10^-170). Mas1p is a subunit of the mitochondrial processing protease (Witte et al., 1988). The protein UMAG_06480 does not have any conserved domains. Moreover, the protein can only be found in smut fungi such as U. bromivora (Similarity 78%, E-value 7x10^-95), S. reilianum (Similarity 83%, E-value 1x10^-85) and U. hordei (Similarity 80%, E-value 8x10^-80).

The protein UMAG_06089 (Fig 3.43, upregulated in planta) was only present in the treated Spp1-GFP and Spp1^D279A-GFP conditions. BLASTp analysis of UMAG_06089 predicted a conserved microsomal signal peptidase 25 kDa subunit (SPC25) domain (E-value 3.04x10^-38), which is part of the signal peptidase complex. The gene is a member of the UPR core genes (Fig 3.10), which is essential for U. maydis (Chapter 3.3.1)

The protein UMAG_02578 (Fig 3.43, not regulated in planta) was only detectable in conditions with the catalytically inactive Spp1^D279A-GFP mutant strain. In a BLASTp analysis of the protein sequence, a luminal heterokaryon incompatibility protein (Het-C) domain was predicted (E-value 0) that is related to het-C of Neurospora crassa (Similarity 53%, E-value 4x10^-140) as well as TinC of A. nidulans (Similarity 60%, E-value 4x10^-136). The protein in U. maydis has a predicted transmembrane domain (Phobius) and a larger cytosolic domain (241 AA), which is only present in smut fungi such as U. bromivora, S. reilianum and U. hordei.

Figure 3.43: Heat map of potential Spp1 interaction partners identified by LC-MS analysis. For LC-MS analysis, the strains SG200∆spp1 Potef:spp1-GFP, SG200∆spp1 Potef:spp1^D279A-GFP and SG200 Potef:GFP-3xHA were grown in CMG to an OD600 of 0.35 and treated 4 h with or without TM (5 µg/ml f.c.) for UPR induction.

Samples were taken and prepared protein extracts were subjected for immunoprecipitation with magnetic agarose GFP-trap beads (Chromotek). Proteins were eluted and subjected to LC-MS analysis. Data analysis was performed with MaxQuant (https://maxquant.org) 1.6.0.16 (parameter file in Appendix File 2) using the label-free quantification method. For statistical analysis of the MaxQuant output, the Perseus (1.6.2.3) framework was used.

The heat map was generated in Perseus by filtering towards proteins being detected in the treated Spp1^D279A-GFP condition. The GFP-3xHA control was used as a negative control, to decrease the number of specific or non-interacting proteins. The LC-MS analysis was performed in three independent replicates, each indicated as a column in the heat map. Values for the proteins (rows) and the conditions (columns) are colored based on the protein abundance, in which high and low log2 LFQ (label-free quantification) intensity values are depicted in red and yellow, respectively. The range of log2 LFQ intensity values is indicated in the color bar shown below. Sample types (Spp1-GFP, Spp1^D279A-GFP) and treatment conditions (without tunicamycin [-TM], with tunicamycin [+TM]) are indicated at the top. Gene regulation of the identified proteins in planta (Lanver et al., 2018) is depicted for 2, 4 and 6 days after inoculation (dpi). Green, gray and red boxes indicate upregulation, the absence of differential gene expression and downregulation, respectively. Data generated in (Hach, 2018).

To examine if UMAG_02578 is involved in processes mediated by Spp1, subcellular localization was examined and deletions strains of UMAG_02578 were tested in a plant infection assay. UMAG_02578

64 However, after UPR activation, a more prominent signal in the nucleus can be observed (Fig 3.44, +TM, white arrows).

Figure 3.44: UMAG_02578-GFP accumulates upon ER stress at the nucleus of U. maydis. The strain SG200 Potef:UMAG_02578-GFP was grown in CMG to an OD600 of 0.8 and were treated with or without TM (5 µg/ml f.c.) for 2 h at 28°C to activate the UPR. DIC = differential interference contrast. Exposure time of GFP channel was set to 350 ms. Scale bar = 20 µm. Data generated in (Hach, 2018).

An infection assay was performed to address the question if UMAG_02578 is essential for pathogenicity.

To this end, deletion mutants of UMAG_02578 were generated. The strains SG200 (WT) and the derivatives ∆UMAG_02578#1, ∆UMAG_02578#3 and ∆UMAG_02578#5 were grown in YEPSlight to an OD600 of 1 and inoculated into 7-day-old maize seedlings. However, all of the UMAG_02578 deletion mutants are indistinguishable from the wildtype strain (Fig 3.45). The function of the protein interaction between Spp1 and UMAG_02578 remains unclear and may be elucidated in the future.

Figure 3.45: Deletion mutants of UMAG_02578 have no impact on pathogenicity. U. maydis strain SG200 (WT) and derivative were inoculated into 7-day-old maize seedlings. Disease symptoms were rated 8 dpi and grouped into categories depicted on the right. n represents the total number of inoculated plants.

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