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Genes to Cells (2009) 14, 205-215. doi:10.1111/j.1365-2443.2008.01263.x
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O-Mannosylation is required for the solubilization of heterologously expressed human β-amyloid precursor protein in Saccharomyces cerevisiae

 Akiko Murakami-Sekimata1a, Ken Sato2b, Ken Sato2c, Akihiko Takashima1 and Akihiko Nakano2,3,*

1 Laboratory of Alzheimer's Disease, RIKEN Brain Science Institute, Wako, Saitama, Japan
2 Molecular Membrane Biology Laboratory, RIKEN Advanced Science Institute, Wako, Saitama 351-0198, Japan
3 Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
In an attempt to express human β-amyloid precursor protein (APP) in yeast, we fortuitously found that this protein is only O-glycosylated in yeast. APP was effectively expressed in yeast, processed by yeast {alpha}-secretases, members of the Yapsin family, to produce N-terminal (sAPP{alpha}) and C-terminal (CTF{alpha}) domains, when its signal sequence was replaced by that of the yeast {alpha}-mating factor. APP is known to acquire N- and O-glycosylation through the endoplasmic reticulum (ER) and the Golgi apparatus and is transported to the plasma membrane in mammalian cells. In spite of the presence of canonical N-glycosylation consensus sequences, APP was not N-glycosylated in the yeast system. Pulse-chase experiments demonstrated that APP received only O-mannosylation in yeast. Examination of yeast pmt mutants, which are defective in the initiation of O-mannosylation in the ER, revealed that Pmt4p is most responsible for the oligosaccharide modification of APP. Maturation of APP was slowed down and aggregated forms of APP were observed by sucrose density gradient fractionation of the {Delta}pmt4 mutant lysate. This caused decreased production of CTF{alpha}. We conclude that O-mannosylation is required for the solubilization of exogenously expressed human APP.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Alzheimer's disease is characterized by the presence of deposit of the amyloid β peptide (Aβ) in the brain (Hardy & Selkoe 2002). The Aβ peptide is produced by proteolytic cleavage of the β-amyloid precursor protein (APP), a type-I transmembrane glycoprotein (Nunan & Small 2000). The Aβ region of APP comprises a sequence of 39–43 amino acid residues located partly within the ectodomain and partly within the transmembrane domain of APP (Fig. 1A). APP is cleaved by three types of proteases designated {alpha}-, β- and {gamma}-secretases. The process by β- and {gamma}-secretases is called the amyloidogenic pathway. The non-amyloidogenic pathway comprises processing by {alpha}-secretase. These two pathways are considered to compete with each other under physiological conditions. APP is subject to a variety of modification and processing during its transport through the secretory pathway. Immature N-glycosylated APP is first synthesized in the endoplasmic reticulum (ER), and then APP matures by acquiring N- and O-glycosylation through the Golgi apparatus; mature APP is transported to the plasma membrane. In the non-amyloidogenic pathway, mature APP is processed by {alpha}-secretase in the late Golgi and the plasma membrane. Previous studies showed that yeast contains functional orthologs of mammalian {alpha}-secretase, the Yapsin family (Yps proteins) (Zhang et al. 1994; Zhang et al. 1997). Yps proteins are GPI-anchored aspartyl proteases and can process human APP at the same site as the mammalian enzyme (Zhang et al. 1994), producing N-terminal (sAPP{alpha}) and C-terminal (CTF{alpha}) domains.


Figure 1
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  		Figure 1  Expression and processing of P{alpha}APP in yeast. (A) Illustration of human APP and its processed C-terminal fragments. P{alpha}APP is a chimeric version of APP, which has its own signal peptide replaced by the yeast {alpha}-factor signal peptide. When APP is processed by {alpha}-secretase (shown by the open triangle), a C-terminal C83 (CTF{alpha}) fragment is produced. The β-secretase processing shown by the closed triangle yields another type of C-terminal fragment. The shaded box is the β-amyloid (Aβ) region and the asterisk is for the {gamma}-secretase site. Peptide sequences recognized by specific antibodies (22C11, 6E10 and anti-APPc) are also shown. TMD, transmembrane domain; Y, N-glycosyl modification site. (B, C) APP was expressed in yeast cells (wild-type strains, YPH500 or BY4742, or deletion strains of Yapsin, {Delta}yps1 or {Delta}yps3) or in mammalian COS7 cells and analyzed by immunoblotting with anti-APPc. Closed arrowheads show the position of the CTF{alpha} fragment, open arrowheads indicate the position of full-length of human APP, and the asterisk points the mature type of human APP in COS7. x, non-specific bands detected by the anti-APPc antibody in COS7 cells; o, degradation products derived from APP. (D) Processed low-molecular-weight bands were characterized by two antibodies, anti-APPc and 6E10. C99 and C83 were expressed in COS7 cells as controls of the C-terminal fragments.

 
Protein O-mannosylation is an evolutionarily conserved protein modification of fundamental importance in many eukaryotes (Endo, 1999; Strahl-Bolsinger et al. 1999). In fungi, the attachment of O-linked mannosyl residues to secretory proteins is essential for cell viability (Gentzsch & Tanner 1996). The impairment of O-mannosylation affects stability, localization, degradation of unfolded proteins, and proper functions of individual proteins (Sanders et al. 1999; Harty et al. 2001; Lommel et al. 2004; Nakatsukasa et al. 2004; Proszynski et al. 2004; Hirayama et al. 2008). Furthermore, aberrant O-mannosylation can interfere with the retrograde transport of misfolded proteins across the ER membrane (Harty et al. 2001). In Saccharomyces cerevisiae, a total of seven PMT family members (Pmt1-7p) are involved in the initiation of O-mannosylation in the ER. After initiation, the O-mannosyl chain is further developed in the Golgi apparatus by other O-mannosyltransferases (Mnt1p, Ktr1p, Ktr3p and Mnn1p etc.). Phylogenetic analyses indicate that the protein O-mannosyltransferases fall into three major groups, PMT1, PMT2 and PMT4 subfamilies (Girrbach et al. 2000; Willer et al. 2002; Girrbach & Strahl 2003). PMT1 and PMT2 subfamilies are highly redundant (Girrbach et al. 2000; Girrbach & Strahl 2003). PMTs show substrate specificities (Gentzsch & Tanner 1997), but the molecular mechanisms of substrate recognition remain unclear. Orthologs of the PMT families are also found in Drosophila, human and mice. Recently, O-mannosyl glycosylation has been shown to represent a new path-mechanism for muscular dystrophy and neuronal migration disorders in human (Beltran-Valero de Bernabe et al. 2002). Although a growing number of papers discuss the roles of O-mannosylation, the common mechanisms that PMT family O-mannosyl transferases identify and modify their substrates and the physiological roles of O-mannosylation are still obscure.

We have been trying to construct the yeast–human heterologous system of APP biogenesis, taking the advantage of the vast knowledge of the yeast secretory pathway to understand the condition of the APP processing. In parallel to our initial purposes, we decided to pursue the interesting finding that human APP is O-glycosylated but not N-glycosylated in yeast to understand the roles of glycosylation on this protein. In this article, we show that, among the PMT family, Pmt4p is most responsible for initiating O-mannosylation of APP, and suggest the role of O-mannosylation in the solubilization of exogenously expressed proteins.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Expression and processing of APP in yeast cells

APP695 is a type-I membrane protein and is highly expressed in the nervous system (Palmert et al. 1989). It has a signal sequence at the N-terminus of the protein. We first tried to make a system to express the authentic APP gene in S. cerevisiae, but it resulted in a very low efficiency. We then replaced the signal sequence of APP695 by that of the yeast {alpha}-mating factor, the pre-pro {alpha}-factor signal peptide sequence, which strikingly improved the expression level of APP in yeast. We named this chimeric gene, P{alpha}APP (Fig. 1A).

The human APP695 is processed by {alpha}-secretase, leading to the production of N-terminal (sAPP) and C-terminal (CTF{alpha}) fragments (Fig. 1A). A typical processing pattern in COS7 cells is shown in Fig. 1B, in which the 110-kDa mature form of APP (asterisk), the 97-kDa immature form (open allow head), and the 10-kDa CTF{alpha} are observed. In wild-type yeast cells (YPH500), the P{alpha}APP protein was also processed yielding a low-molecular-weight band similar to CTF{alpha} (Fig. 1B). The yeast orthologs of {alpha}-secretase, Yps proteins (Yps1, Yps2 and Yps3) (Zhang et al. 1994; Zhang et al. 1997), were expected to process human APP in a redundant manner. As shown in Fig. 1C, {Delta}yps1 and {Delta}yps3 showed low efficiency in the production of the 10-kDa CTF{alpha}-like band, suggesting that they are indeed responsible for the processing of APP. A similar defect was also observed with {Delta}yps2 (unpublished results, A. Murakami-Sekimata). To investigate the nature of this 10-kDa CTF{alpha}-like band produced in yeast, we examined its reactivity with a monoclonal antibody specific to the β-processed end of the APP (6E10; see Fig. 1A). As a control, the expression of APP695 in COS7 cells led to the production of C83 (CTF{alpha}), which was detected by the antibody against the C-terminus of APP (APPc) but not by the 6E10 antibody (Fig. 1D). The control fragment C99 was recognized by either of the two antibodies, 6E10 and APPc. Thus we concluded that the processing occurring in yeast by Yps proteins is similar to that in mammalian cells.

To examine the further fate of processed APP in yeast, we analyzed materials secreted to the periplasm by immunoblotting with two monoclonal antibodies, 22C11 and 6E10. 22C11 is specific to the N-terminal ectodomain of APP (Fig. 1A). It was previously shown that the secreted form of APP (sAPP) is not released to the medium but is trapped in the periplasmic space of yeast (Hines et al. 1994) and a DTT treatment is required to extract sAPP from the cell surface (Komano et al. 1999). The DTT extract from the cells was concentrated and subjected to immunoblotting (Fig. 2A, sup). The band of approximately 90 kDa was detected by the 22C11 and 6E10 antibodies, but not by the APPc antibody. The full-length APP was detected in the cell lysate by the three antibodies (Fig. 2A, ppt). When APP was expressed in a temperature-sensitive secretion mutant, sec12-4, which has a defect in the ER-to-Golgi transport, CTF{alpha} was not produced at the restrictive temperature (37 °C, Fig. 2B). These results suggest that APP is transported through the yeast secretory pathway and processed by yeast {alpha}-secretases in the Golgi apparatus or later compartments.


Figure 2
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  		Figure 2  Secretion of the N-terminal fragment of APP. (A) Secretion of the N-terminal fragment of APP was analyzed by immunoblotting with indicated antibodies. Yeast cells expressing APP were treated with DTT to extract secreted materials (sup) and then lysed (ppt). For details, see under Materials and Methods. Asterisks, small amounts of full-length APP extracted from the plasma membrane by DTT treatment. (B) Pulse-chase analysis of APP in sec12-4 mutant cells. The cells labeled with [35S] were chased for indicated times at 23 °C or the restrictive temperature of sec12-4, 37 °C, and subjected to immunoprecipitation with anti-APPc. The graph indicates the amounts of CTF{alpha} produced as expressed by the ratio over total (full length plus CTF{alpha}).

 
Glycosyl modification of APP in yeast

Like in other eukaryotes, two types of oligosaccharide modifications can occur in yeast: O-glycosylation and N-glycosylation, both being initiated in the ER and extended in the Golgi. To examine the properties of the possible glycosylation of APP in yeast, we carried out a pulse-chase and immunoprecipitation analysis with antibodies specific for {alpha}1,6- and {alpha}1,3-mannosyl linkages (Sato et al. 1996). For the yeast N-glycosylation, it has been established that the anti-{alpha}1,6-mannosyl antibody recognizes modifications acquired in the cis-Golgi and the anti-{alpha}1,3-mannosyl antibody detects those in the medial Golgi. Carboxypeptidase Y (CPY), a vacuolar glycoprotein, is a well-known marker of the yeast vesicular traffic pathway. When CPY was immunoprecipitated by the anti-CPY antibody and then by the mannosyl linkage specific antibodies (Fig. 3A), the modification and maturation of the molecular species were observed first from the non-reactive p1 (ER) form, via the p2 (Golgi) form that is recognized by both {alpha}1,6- and {alpha}1,3-mannosyl antibodies, and then to the mature (m) form during the 15-min chase time. In a parallel experiment (Fig. 3B), we were able to detect only {alpha}1,3-mannosyl linkage on the APP expressed in yeast. A similar experiment was repeated in the presence of tunicamycin, a reagent that completely blocks the N-linked modification (Fig. 4A). The {alpha}1,3 linkage was still recognized under the condition that CPY was totally non-glycosylated (compare to the right panel). The presence of non-N-lined glycosylation on APP was also confirmed by a concanavalin A (ConA)-Sepharose binding experiment. Again in the presence of tunicamyin, under which condition unglycosylated CPY (unbound to ConA) was detected by immunoblotting, APP was still totally bound to ConA (Fig. S1 in Supporting Information). The {alpha}1,3-linkage on APP was observed when APP was expressed in sec7-1, a mutant blocked in the Golgi, but not in sec18-1 or sec12-4 mutants (Fig. 4B), which are defective in transport from the ER. These results strongly suggest that the {alpha}1,3-mannosyl modification on APP is due to the O-linked {alpha}1,3-mannosyl linkage that was added in the Golgi apparatus.


Figure 3
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  		Figure 3  Glycosyl modification of APP. Yeast cells expressing APP were [35S] labeled and chased for indicated times, lysed and subjected to the first immunoprecipitation with anti-APPc, and then the second immunoprecipitation with antibodies against {alpha}1,6- and {alpha}1,3-mannosyl linkages (B). Glycosyl modification of carboxypeptidase Y (CPY) was shown as a control (A). p1, ER form; p2, Golgi form; m, mature form.

 

Figure 4
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  		Figure 4  Glycosyl modification of APP in yeast is O-linked mannosylation. (A) After treatment of APP-expressing yeast cells with 10 µg/mL tunicamycin, a pulse-chase experiment was carried out as in Fig. 3. The result for CPY as a control with (+) or without (–) tunicamycin is shown for the 15-min chase time. ng, non-glycosylated form. (B) The same pulse-chase analysis on APP was carried out in secretion mutants. sec18-1 and sec12-4 block the transport from the ER to the Golgi, and sec7-1 arrests the export from the Golgi, at the restrictive temperature 37 °C.

 
PMT genes are responsible for O-mannosylation of APP in yeast

In yeast, O-mannosylation initiates in the ER and continues in the Golgi. For the {alpha}1,3-mannosylation on the O-linked chains, O-mannosyltransferases encoded by PMT genes are known to be responsible (Strahl-Bolsinger et al. 1999). We examined the modification of APP in the pmt mutants. As shown in Fig. 5, different degrees of deficiency in {alpha}1,3 modification were observed in {Delta}pmt1–{Delta}pmt6 mutants. No such differences were found in the case of control CPY. Numbers shown under the lanes of {alpha}1,3 immunoprecipitation indicate the proportion of APP that acquired the {alpha}1,3-mannosyl linkage to the total amount of APP. {Delta}pmt4 was most defective, {Delta}pmt1 and {Delta}pmt2 were also affected, whereas others were rather normal.


Figure 5
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  		Figure 5  O-Mannosyl modification of APP in pmt mutants. Pulse-chase analysis for investigating O-mannosyl modification of APP was carried out in {Delta}pmt mutants. The numbers shown under the lanes of {alpha}1,3 antibody indicate the proportion of APP modified with {alpha}1,3-mannosyl linkage (% of total APP). CPY was used as a control.

 
Correlation between O-mannosylation of APP and CTF{alpha} production

To investigate the effect of O-mannosylation on APP processing in yeast, the production of CTF{alpha} was examined in the pmt mutants. Three independent experiments were carried out and quantified (Fig. 6). As in the case of O-mannosylation, {Delta}pmt1, {Delta}pmt2 and {Delta}pmt4 showed significant defects in the production of CTF{alpha}. The {Delta}pmt4 also showed a shift in migration of the full-length APP (top panel). These results suggest a correlation between O-mannosylation and the processing of APP in yeast.


Figure 6
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  		Figure 6  CTF{alpha} production in pmt mutants. The processing of APP was examined by immunoblotting in {Delta}pmt mutant cells. The full-length APP and CTF{alpha} was detected on the same membrane with the anti-APPc antibody. The lower panel indicates the quantified result of the CTF{alpha} production as expressed in % total. The error bars show standard deviations calculated from three independent immunoblots. *P < 0.05 compared with the result of the wild type (BY4742) (Student's t-test).

 
Because the amount of CTF{alpha} detected by immunoblotting was lower in {Delta}pmt4, we examined the stability of CTF{alpha} by a pulse-chase analysis in {Delta}pmt4 to test whether the decrease is due to accelerated turnover or decreased processing. The full-length APP was more stabilized in {Delta}pmt4 cells than in wild-type cells (BY4742, Fig. 7A). For CTF{alpha}, the rates of turnover did not differ significantly between {Delta}pmt4 and wild-type cells (Fig. 7B), suggesting that the lower CTF{alpha} production is not because of the reduced stability. Next, we carried out a pulse-chase experiment with shorter chase times (0–15 min) to examine the rate of CTF{alpha} production. The pulse-chase experiment showed a small but reproducible delay in the {Delta}pmt4 mutant (Fig. 7C). These results suggest that the decrease of CTF{alpha} amount was caused by the delay of processing, not by accelerated turnover.


Figure 7
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  		Figure 7  Detection of degradation of full-length P{alpha}APP and production of the CTF{alpha} by pulse-chase experiments. Life times of full-length (A) or CTF{alpha} (B) were tested by pulse-chase experiment in wild-type (BY4742) or {Delta}pmt4. Pulse time was 5 min and chase time was 0–240 min. The results were quantified and, shown in graphs. The vertical line shows percentage of amount of CTF{alpha} or full-length in logarithmic phase. For the quantification, the result on 0 min is considered 100%. Result of chase during early time is shown in (C) for 0–15 min. For this experiment, pulse-time was 3 min. The maximum amount of CTF{alpha} is 100% in the vertical line.

 
To obtain an insight into the role of O-mannosylation in APP processing, we tried to examine the folding status of APP in wild-type and {Delta}pmt4 yeast cells. Triton-X-100 lysates were prepared and subjected to a sedimentation analysis through a sucrose density gradient. The amount of full-length APP was examined by SDS-PAGE and immunoblotting for each fraction of the gradient, and the results are shown as % total in Fig. 8. In the case of wild-type cells, an appreciable proportion of APP distributed for fractions 1 to 8, which probably represent the monomers or small oligomers of APP. By a sharp contrast, in {Delta}pmt4, the major fraction of APP was recovered at the bottom of the gradient. Basically the same results were obtained in three independent experiments, and thus we concluded that the deletion of PMT4 promoted the aggregation of APP. The aggregation of APP was also observed in wild-type yeast cells, but the extent was approximately half.


Figure 8
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  		Figure 8  APP shows more tendency to form aggregates in the {Delta}pmt4 mutant. Wild-type (BY4742) and {Delta}pmt4 cells were disrupted by glass-beads agitation and Triton X-100 treatment, and then subjected to sucrose density gradient centrifugation. Fractions were collected and analyzed by immunoblotting with the anti-APPc antibody. Molecular weight marker proteins were layered on the same sucrose gradient and centrifuged at the same time, and the positions of the marker proteins are shown at the top of the panel. Markers: 25 kDa, chymotrypsin; 67 kDa, albumin; 158 kDa, aldolase; 440 kDa, ferritin.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
When APP is expressed in yeast, it is proteolytically processed by Yps proteins, the yeast orthologs of {alpha}-secretases (Zhang et al. 1994; Zhang et al. 1997). APP is processed effectively in the wild type, but not in {Delta}yps1-3 mutant cells. The processing appears to occur in the Golgi and the plasma membrane, because the block of protein traffic from the Golgi to the plasma membrane severely affects the processing, and the arrest in the ER completely abolishes it (Fig. 2B). The most unexpected but interesting finding in this work is that APP is not N-glycosylated and only O-mannosylated in the transport process. (i) APP produced in the presence of tunicamycin is still able to bind ConA (Fig. S1). (ii) N-glycosylated proteins in yeast acquire both {alpha}1,6- and {alpha}1,3-mannosyl linkages, but we detect only {alpha}1,3-mannosyl modifications on APP, which is resistant to tunicamycin treatment (Figs 3 and 4A). (iii) The {alpha}1,3-mannosyl linkage occurs in the Golgi apparatus as revealed by pulse-chase experiments with secretion mutants (Fig. 4B). (iv) The initiation of O-glycosylation takes place in the ER by the action of protein O-mannosyltransferases (PMTs). The {alpha}1,3-mannosyl linkage of APP is not detected in {Delta}pmt4. These results indicate that heterologously expressed human APP acquires O-mannosylation instead of N-glycosylation in yeast, in spite of the presence of canonical consensus sequences for N-glycosylation.

The sucrose-density-gradient centrifugation experiment shows that APP is aggregated in an O-mannosylation deficient-mutant, {Delta}pmt4. The processing of APP that leads to the production of CTF{alpha} is delayed in {Delta}pmt4. These results suggest that O-mannosylation is required for keeping APP soluble.

Pmt family has substrate specificity

The PMT proteins are reported to show substrate specificity (Gentzsch & Tanner 1997). Indeed, five proteins tested in the present work (chitinase, {alpha}-agglutinin, Kre9p, Bar1p, and Pir2p/hsp150) are mainly underglycosylated in {Delta}pmt1 and {Delta}pmt2 mutants. Two of O-mannosylated proteins, Ggp1p/Gas1p and Kex2p, are not at all affected in {Delta}pmt1 and {Delta}pmt2 mutants but are clearly underglycosylated when PMT4 is mutated. The PMT3 mutation affects O-mannosylation of chitinase only in the genetic background of a pmt1pmt2 double mutation, indicating that Pmt1p and Pmt2p complex can compensate for a deleted PMT3. Pmt proteins (Pmt1-7p) comprise at least three subfamilies. Members of the PMT1 subfamily (Pmt1p and Pmt5p) interact in pairs with members of the PMT2 subfamily (Pmt2p and Pmt3p). Pmt1p-Pmt2p and Pmt5p-Pmt3p are the predominant complexes present in wild-type cells. Pmt4p is the single member of the PMT4 subfamily; it makes homodimers and shows no redundancy (Girrbach & Strahl 2003). We have demonstrated that APP has a clear dependence on Pmt4p and is less but significantly affected if Pmt1p or Pmt2p is depleted. PMT1, PMT2 subfamilies are redundant and a single deletion of them can be compensated. Consensus sequences of O-glycosylation are not clear. The O-mannosylation of APP has dependence on Pmt4p and to some extent on Pmt1p-Pmt2p. Perhaps human APP contains a recognition structure, which Pmt4p favors (Hutzler et al. 2007). Ecker et al. (2003) reported that the O-mannosylation by Pmt4p prevents N-glycosylation in Ccw5p. They proposed that O- and N-glycosylation machineries compete for Ccw5p. If a similar event happened on APP, both {alpha}1,6 and {alpha}1,3 might have been detected in {Delta}pmt4, in which O-mannosylation is reduced. Hirayama et al. (2008) have recently shown that Pmt1p and Pmt2p are important for proteasome-dependent degradation of Gas1*p and the degradation is altered from a primarily proteasome-dependent pathway to a vacuolar proteases-dependent pathway in the {Delta}pmt1{Delta}pmt2 background. The roles of Pmt1p-Pmt2p in the fate of APP expressed in yeast would also be an interesting next question to be addressed.

Deficiency in O-glycosylation causes aggregation and stabilization of the full-length APP

O-glycosylation has been discussed as a sorting determinant for cell surface delivery (Proszynski et al. 2004). There are also reports that O-glycosylation affects protein degradation; some proteins become unstable when O-mannosylation is deficient in {Delta}pmt1 and {Delta}pmt4 mutants (Sanders et al. 1999; Harty et al. 2001; Lommel et al. 2004). Although the degradation rate of full-length APP and CTF{alpha} did not differ much between the wild type and {Delta}pmt4, we have shown that APP is highly aggregated and its processing is retarded in {Delta}pmt4. We have concluded that O-mannosylation by Pmt4p is required for APP solubilization and further processing. It is yet unclear whether the O-mannosylation deficiency causes the transport problem or the aggregation problem occurs first. If APP in {Delta}pmt4 is not transported effectively to the plasma membrane through the Golgi, APP would be accumulated and aggregated. Alternatively, APP may be aggregated first, which then impairs normal transport and processing of the protein. Other possibilities also remain, for example, the defects of O-mannosylation might indirectly affect the processing. The promoted aggregation and reduced processing of APP in the {Delta}pmt4 mutant suggest that APP needs Pmt4p-dependent O-mannosylation in yeast to avoid aggregation and ensure effective transport and processing by {alpha}-secretase.

Orthologs of PMT family in higher eukaryotes

In higher eukaryotes, O-type glycosylation links a variety of sugar moiety to Thr/Ser residues of proteins, such as fucose, glucose, N-acetylglucosamine, xylose, galactose and mannose (Spiro 2002). O-mannose-type glycosylation used to be regarded specific to yeast before. However, orthologs of the PMT4 subfamily members have lately been identified in Drosophila meranogaster (rotated abdomen, rt) and human (hPOMT1) (Martin-Blanco & Garcia-Bellido 1996; Jurado et al. 1999; Willer et al. 2003). Drosophila rt mutants feature reduced viability as well as pronounced defects in muscle development, indicating an essential role of PMTs in not only lower but also higher eukaryotes. Human hPOMT1 is ubiquitously expressed and higher levels of expression are observed in fetal brain, adult pituitary and testis (Jurado et al. 1999). It has been estimated that 30% of all O-linked sugar chains in brain are O-mannose based (Chai et al. 1999). Importantly, hPOMT1 is also implicated in a human disease, Walker–Warburg Syndrome (Jurado et al. 1999). Walker–Warburg Syndrome is a recessive disorder characterized by severe brain malformations, muscular dystrophy, and structural eye abnormalities. Moreover, POMT2, the ortholog of the PMT2 subfamily have also been found in human, mouse and Drosophila (Willer et al. 2002). POMT2 is predominantly expressed in the testis tissue. Human endogenous APP is known to be O-glycosylated, but the sugar components of its oligosaccharide chains remain to be examined, and it is not known whether hPOMT1 and POMT2 work as enzymes for human APP. To address such emerging possibilities on the important roles of O-mannosylation by PMTs, more studies will be necessary, for example, on the molecular mechanisms of substrate identification.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Yeast strains

The yeast strains used in this study are shown in Table 1. Cells were grown at 30 °C in YP medium (2% (w/v) polypeptone, 1% (w/v) yeast extract) containing 2% glucose (YPD) or in MC medium (0.67% (w/v) yeast nitrogen base without amino acids, and 0.5% (w/v) casamino acids) containing 2% (w/v) glucose (MCD) supplemented appropriately. For Saccharomyces Deletion Project's strains, G418 was supplemented in YPD at the concentration of 200 µg/mL.


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  		Table 1  List of strains used in this study
 
Plasmids and construction

pTU1 (pRS316 derived, CEN, TDH3 promoter) (Ueda et al. 2001) was used for APP expression. First, the open reading frame of APP695 was amplified with primers containing EcoRI sites by the polymerase chain reaction (PCR). The product was digested by EcoRI and inserted into the EcoRI sites of pTU1 (pTU1-APP). Next, a DNA fragment encoding the signal peptide sequence of yeast {alpha}-mating pheromone (MRFPSIFTAVLFAASSALAAPV) was amplified with primers [(5'-GAAGATCTATGAGATTTC CCTTCAATTTTTACTGCAGTT-3') and (5'-GGGTACCTC GACTGGAGCAGCTAATGCGGAGGA-3')]. The product was digested by BglII and KpnI and cloned into BglII and KpnI sites of pTU1 (pTU1-p{alpha}). Second, APP with the CMK1 terminator was withdrawn from pTU1-APP by digestion by KpnI, and the APP fragment was cloned into the KpnI site of pTU1-p{alpha}. The final plasmid was named pTU1-p{alpha}APP and used for P{alpha}APP expression.

Detection of secreted N-terminal APP by immunoblotting

Fifty milliliters of yeast culture was harvested and incubated with 10 mL of 0.01 M DTT in 0.1 M Tris–HCl (pH 9.4) at 30 °C for 30 min. After centrifugation at 1000 g for 10 min, supernatant was cleared at 40 000 g again and added to one-tenth volume of 100% TCA (final concentration of 10%). Following incubation on ice for 1 h, the precipitate was collected by centrifugation at 15 000 g for 15 min and washed twice with 100% ethanol. The precipitate was dried up and resuspended in 45 µL of SDS-sampling buffer (Fig 2C: sup). The cell pellets of the 0.01 M DTT treatment was crushed by agitation with glass beads in 200 µL of 1 x SDS-sampling buffer and the resulting cell lysate was used for SDS-PAGE (Fig 2C: ppt). For immunoblotting, anti-APPc antibody was used for detection of the APP full-length protein and the C-terminal fragment. Monoclonal antibodies 22C11 and 6E10 were used for detection of the APP N-terminal fragment. LAS-1000 (Fujifilm) was used to image the blottings by chemiluminescence.

Pulse-chase analysis for investigating glycosyl modification of APP

Metabolic labeling of yeast cells and preparation of cell extracts were basically carried out as previously described (Rothblatt & Schekman 1989; Nishikawa et al. 1990). For detection of {alpha}1,6- and {alpha}1,3-mannosyl linkages, their specific antibodies were used (Sato et al. 1996). Cells were grown to 1-2 x 107 cells/mL in minimal medium (MCD), harvested and washed once with distilled water. Cells (2 x 107) were incubated in 200 µL of sulfate-free minimal medium at an appropriate temperature and labeled with 1.85 MBq (50 µCi) of Tran-35S labeling mix (L-[35S] methionine and L-[35S] cysteine mix; Amersham Pharmacia Biotech) for 1 or 5 min. Chase was initiated by the addition of 1 mL chase buffer (MCD plus 100 µM each of (NH4)2SO4, L-methionine and L-cysteine). After an appropriate period of time, the incubation was terminated by the addition of 200 µL of cold 60 mM NaN3 (final concentration was 10 mM). Labeled cells were washed with 1 mL of 10 M NaN3 and resuspended in 200 µL of Tris-buffer containing 1% SDS and protease inhibitors mixture. The suspended cells were disrupted by glass beads followed by heating at 95 °C for 5 min. Extracts were adjusted to 1 mL by the addition of Tris-buffer containing 2% of Triton X-100 and protease inhibitors mixture and clarified by centrifugation at 15 000 g for 10 min. The supernatant were incubated with 10 µL of anti-APPc antibody at 4 °C overnight. Forty µL of 50% (v/v) suspension of protein A-Sepharose CL-4B (Sigma) in Tris-buffer was added, and incubation continued with gentle agitation at room temperature for 2 h. The Sepharose beads were collected by centrifugation and washed with buffers described (Rothblatt & Schekman 1989). Immunoprecipitates were eluted from the beads by heating at 95 °C for 5 min in 210 µL of 1% SDS and 1% 2-melcaptoethanol and the eluted immunoprecipitates were divided into three 60 µL aliquots. The aliquots were diluted with 10x volume of Tris-buffer containing 2% of Triton X-100, and subjected to the second immunoprecipitation with anti-APPc, anti-{alpha}1,6- or anti-{alpha}1,3-mannosyl linkage antibodies each. The eluted second immunoprecipitates were analyzed by SDS-PAGE and scanning by imaging analyzer (BAS-2500, by Fujifilm).

Sucrose density gradient centrifugation

Sucrose density gradients were carried out by the method described (Nishikawa et al. 2001) with some modifications. Exponential growing phase of cells (5 x 107) were collected by centrifugation and washed once with ice-cold 10 mM NaN3. Cells were suspended in 600 µL of 10 mM Hepes–KOH, pH 7.4, 2 mM EDTA, 1 mM PMSF and protease inhibitors mix (Sigma), and disrupted by agitation with glass beads. After removal of cell debris by centrifugation, Triton-X-100 was added to the cell lysate up to 1% (final concentration), which was subsequently centrifuged at 12 000 g at 4 °C for 10 min. The cleared lysate was layered onto linear sucrose gradients (4.4 mL, 5–40% of sucrose in 10 mM Hepes–KOH, pH 7.4, 2 mM EDTA, 1 mM PMSF and 0.1% Triton X-100), and centrifuged at 145 000 g at 4 °C for 20 h. After centrifugation, fractions were collected from the top and analyzed by immunoblotting using anti-APP C-terminal antibody.


   Acknowledgements
 
Authors are grateful to Shuh-ichi Nishikawa of Nagoya University for his critical comments, to Yoshitaka Tatebayashi of the Laboratory of Alzheimer's Diseases, RIKEN BSI, and Hironori Higashio of Tokushima University and all members of the Molecular Membrane Biology Laboratory, RIKEN ASI, for constructive discussions. Authors also thank Ohoshi Murayama of Azabu University and Jun-Mi Park of RIKEN BSI for the antibodies against APPc, Takashi Ueda of the University of Tokyo for the pTU1 vector and Sabine Strahl of the University of Heidelberg for {Delta}pmt4 strains. This work was supported by a Grant-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Murakami-Sekimata, A.), and by a President's Special Research Grant of RIKEN (Nakano, A. and Takashima, A.).


   Footnotes
 
Communicated by: Hiroyuki Araki

aPresent address: Department of Health Sciences, Graduate School of Medicine, Tohoku University, 2-1 Seiryo, Aoba-ku, Sendai 980-8575, Japan Back

bPresent address: Laboratory of Molecular Traffic, Department of Molecular Cellular Biology, Institute of Molecular and Cellular Regulation, Gunma University, Maebashi, Gunma 371-8512, Japan Back

cPresent address: Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Meguro-ku, Tokyo 153-8902, Japan Back

* Correspondence: nakano{at}riken.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
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Received: 19 June 2008
Accepted: 9 November 2008




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