SmGPI1 is a dual targeted protein

GPI-anchored proteins normally localize to the outer leaflet of the plasma membrane (Fujita &

Kinoshita, 2012, Singh et al., 2011). In S. cerevisiae, the region ω-1 to ω-5 can support membrane localization (Caro et al., 1997, Frieman & Cormack, 2003, Hamada et al., 1999,

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Stefanova et al., 1991). Moreover, GPI-anchored proteins in ascomycetes can also be covalently bound to the cell wall (Gilbert et al., 2012). The switch between localization to the membrane or cell wall depends among other factors, on the presence or absence of dibasic residues at position ω-1 or ω-2 (Stefanova et al., 1991, Ouyang et al., 2013). Absence of dibasic residues at position ω-1 and ω-2 support localization of the anchored protein to the cell wall (Stefanova et al., 1991, Ouyang et al., 2013). Fluorescence microscopy and Western blot analysis of eGFP-tagged SmGPI1, however, showed localization at the plasma membrane or cell wall, a fluorescence pattern similar to mitochondrial localization and partial secretion (Figure 16, Figure 18 and Figure 19).

A high content of serine and threonine residues in GPI-anchored proteins was shown to support cell-wall attachment of GPI-anchored proteins, approximately 70% of S. cerevisiae cell-wall bound GPI-anchored proteins contain more than 30% serine/threonine residues (Frieman &

Cormack, 2004).

Sequence analysis of SmGPI1 revealed a serine/threonine content of 20% in total, compared to the average in proteins of 7.6% for serine and 6% for threonine (Bruice, 2004), the serine content of SmGPI1 (12.7%) but not the threonine (7.2%) is significantly increased. These results indicate that the final destination of GPI-anchored proteins is mediated by several factors. For example, De Sampaio et al. (1999) showed for the glucanosyltransferase GAS1p in S. cerevisiae localization to the cell wall, although it contains a dibasic residue at position ω-1 and ω-2.

SmGPI1-eGFP localizes to structures, resembling the plasma membrane or the cell wall (Figure 19). Moreover, after differential centrifugation, SmGPI1 was mainly found in the cell detritus, but in case of deletion of the predicted region for GPI-anchor attachment, it appears mainly in the cytosol (Figure 16). The cell detritus contained remnants after early centrifugation, such as parts of the cell wall. Thus, the region containing the ω-residue might be crucial for localization of SmGPI1 to the outer leaflet of the plasma membrane. This data is also supported by eGFP localization of SmGPI1 without the GPI-anchor attachment region (Figure 19), which was no longer present at the cell wall or membrane (Figure 19). These findings are consistent with many studies, made with other organisms. Ouyang et al. (2013) showed recently that only the signal sequence and the region for GPI-anchor attachment comprising ω-10 to the C-terminal end of cell-wall protein Mp1p, glucanosyltransferase Gel1 and Ecm33, which function in maintaining fungal cell-wall integrity and virulence, are sufficient for proper localization to the cell

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membrane or the cell wall. In their experiments, they fused the respective signal sequences and omega regions to eGFP and thus, mediated eGFP localization to the cell wall and membrane. In contrast to De Sampaio et al. (1999), Ouyang et al. (2013) showed that mutation of the residues ω-1 and ω-2 can alter the protein localization. Cryptococcus neoformans chitin deacetylase 2 is an enzyme that converts chitin to chitosan and is an established virulence factor for C. neoformans infection. Gilbert et al. (2012) showed that the protein is bound to membranes and non-covalently associated to cell walls. Interestingly, cell wall association was independent from its GPI-anchor.

SmGPI1 full-length protein also appears in the cell-free medium (Figure 18). Deletion of its omega region not only impairs cell-wall localization, but slightly decreases secretion of the protein. Secretion of GPI-anchored proteins in general is well documented (Low, 1989, Mayor &

Riezman, 2004, Paulick & Bertozzi, 2008). Djordjevic et al. (2005) showed that C. neoformans virulence factor phospholipase B1 localizes (Plb1) to the cell wall, membrane structures and was also detectable in the cell-free medium. Furthermore, that this localization pattern was GPI-anchor dependent. Similar to SmGPI1, deletion of the GPI-GPI-anchor led to an increased secretion of the protein. Moreover, SmGPI1 was shown to localize to structures resembling mitochondria (Figure 19). This was confirmed by co-staining with MitoTracker Red and likely caused the signal detected for the membrane fraction after differential centrifugation (Figure 16). In eukaryotes, dual targeting of a single proteins to more than one subcellular compartment is well documented (Raza, 2011, Ben-Menachem et al., 2011, Dinur-Mills et al., 2008, Yogev et al., 2011). These examples include GPI-anchored proteins such as S. cerevisiae β-1,3-glucanosyltransferase GAS1, which plays as a GPI-anchored cell-wall protein a role in the formation and maintenance of the cell wall and when targeted to the nucleus, in regulation of transcriptional gene silencing and rDNA stability. When targeted to the cell wall, GAS1 elongates and arranges 1,3-glucan side chains, which are linked to glucan, chitin and proteins, and in sum, form the main layer of cell walls. The sub population of GAS1 detected in the nucleus interacts with the histone deacetylase SIR2 and increases rDNA silencing in a SIR2-dependent manner (Koch & Pillus, 2009, Bauer et al., 2014). Pfeiffer et al. (2013) demonstrated that ER signal peptides of the GPI-anchored prion-like Shadoo, the neuropeptide hormone somatostatin, and the amyloid precursor protein mediate alternative targeting to mitochondria.

This effect is mediated by structural features within the nascent chain; the signal sequences of 114

each protein promotes proper ER import of the nascent chain containing alpha-helical domains, but unstructured polypeptides are targeted to mitochondria. Increased transport to mitochondria causes unproductive transport to the ER lumen, and vice versa. By this, they presented a novel mechanism of dual targeting of proteins to the ER or mitochondria, facilitated by structural features in the nascent chain.

In silico analysis using GlobPlot2 (Linding et al., 2003) revealed extended intrinsic disorder regions within SmGPI1 (Figure 49). The signal sequence of SmGPI1 seemed to be necessary for mitochondrial localization because N-terminally eGFP-tagged SmGPI1 (data not shown) and SmGPI1 with its first 27 aa deleted led to a diffuse localization to the cytosol. In contrast, removal of the C-terminal GPI-signal sequence slightly increased mitochondrial localization (Figure 19).

Figure 49. SmGPI1 exhibits regions of disorder. Shown is the protein precursor aa 1-253. The omega residue is marked in black. Grey boxes and italic letters in the aa sequence below represent N-terminal and C-terminal signal sequences. Arrows and grey capital letters in the aa sequence below represent disorder regions predicted by GlobPlot2 (Linding et al., 2003).

Similarly, Pfeiffer et al. (2013) showed that loss of the C-terminal GPI-signal sequence interferes with efficient ER import and increased mitochondrial import of mammalian GPI-anchored prion proteins. In this study was shown, that the C-terminal alpha-helical structure of Shadoo, which lies within the region for GPI-anchor attachment, can mediate ER import of the intrinsically disordered protein. In silico analysis with Quick_2D (Biegert et al., 2006) predicted for SmGPI1

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a single alpha-helical structure at the C-terminus. However, deletion of the C-terminal signal sequence of SmGPI1 did not completely impaired secretion but changed localization to the cell wall as shown by differential centrifugation and fluorescence microscopy (Figure 16 and Figure 19). The summarized mechanism of dual targeting of SmGPI1 in S. macrospora is shown in Figure 50.

Figure 50. Dual targeting of SmGPI1 in S. macrospora. Features within the nascent chain determine the destination of SmGPI1, which is targeted to the cell wall and the mitochondria. Alpha-helical regions within the nascent chain facilitate transport to the ER and inhibit targeting to the mitochondria.

Unstructured regions within the nascent chain regulate this process vice versa. For localization to the cell wall, the protein precursor is processed in the ER and transported via vesicles to the final destination.

Based on the model presented by Pfeiffer et al. (2013).

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