1 Department of Molecular Biology and Genetics and Program in Cellular, Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore, MD 21205, USA
2 Department of Molecular Medicine, Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore, MD 21205, USA
Correspondence
Brendan P. Cormack
bcormack{at}bs.jhmi.edu
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ABSTRACT |
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INTRODUCTION |
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While the majority of GPI proteins in S. cerevisiae are thought to be GPI-CWPs (incorporated into the cell wall), there are some that are largely retained at the plasma membrane. In several instances, this difference in plasma membrane or cell wall localization has been shown to be important for function. For the protein Ecm33p, correct localization of the GPI protein to the membrane is important for viability. If Ecm33p is mis-localized to the cell wall by changing its GPI anchor to that of a GPI-CWP, the cells are non-viable (Terashima et al., 2003). In experiments from our own lab, we found that optimal function of the C. glabrata Epa1p adhesin also depends on the nature of the GPI anchor signal. Chimeric constructs in which the ligand-binding domain of Epa1p was fused to GPI signal sequences were fully functional to mediate adherence when the fusions were made to GPI anchor signals from GPI-CWPs, but were significantly compromised for function when the fusions were made to GPI anchor signals from GPI plasma membrane proteins (GPI-PMPs) (Frieman & Cormack, 2003
).
How does the cell recognize two classes of GPI proteins and ensure that GPI-PMPs remain primarily membrane-associated while GPI-CWPs are removed from the plasma membrane, eventually to be cross-linked to glucan? Klis proposed, based on bioinformatic considerations, that GPI-PMPs contain at least two basic amino acids in the region upstream of the site, while GPI-CWPs do not have such a dibasic motif (Caro et al., 1997
). There is considerable experimental support that the dibasic motif does act to retain GPI proteins at the plasma membrane. Using fusions of the GPI signal sequences from S. cerevisiae to
-galactosidase, Hamada et al. (1998)
found a good correlation between presence or absence of the dibasic motif and partitioning of the fusion protein to the plasma membrane or cell wall. Analysis of various point mutations in specific GPI anchor signal sequences also supported the importance of the dibasic motif in GPI protein localization. Experiments from our own lab also support the overall importance of the amino acids upstream of the
-proximal site in GPI protein targeting. If a GPI-CWP, such as Cwp2p, was mutated to carry a dibasic motif preceding the
site, it no longer trafficked to the cell wall, but was retained at the plasma membrane. Conversely, if we mutated the dibasic motif in a model GPI-PMP to other amino acids, the mutant protein was efficiently targeted to the cell wall (Frieman & Cormack, 2003
).
In contrast to these studies, other work suggests that the dibasic motif model for GPI protein distribution may be too simplistic (De Sampaio et al., 1999). Importantly, this paper showed that Gas1p or a fusion protein between
-galactosidase and the GPI signal of Gas1p were found primarily in the cell wall even though, according to the dibasic motif model, these should have been localized primarily to the plasma membrane. This result, combined with the demonstrated role of the
-proximal site in determining distribution of protein between the plasma membrane and cell wall (Frieman & Cormack, 2003
; Hamada et al., 1998
), suggests that more than one signal in the protein may impact on the ultimate distribution of GPI proteins in the cell.
In the current paper we begin to define a second signal that affects the distribution of GPI proteins between membrane and cell wall, and which in fact is able to override the -proximal signal. We find that amino acid stretches rich in serine and threonine residues promote localization to the cell wall even in proteins whose
-proximal signal contains a dibasic motif (and which therefore would be expected to localize to the plasma membrane). These data show that the cell can integrate multiple sequence signals in determining the ultimate distribution of GPI proteins between cell wall and cell membrane.
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METHODS |
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Transformation of S. cerevisiae.
Transformation of S. cerevisiae was carried out using the modified lithium acetate protocol (Gietz et al., 1992). Cells were grown in YPD to early exponential phase and collected by centrifugation. They were washed twice with water and resuspended in 0·1 M lithium acetate, 1 mM EDTA and 10 mM Tris, pH 7·5. Transforming DNA (1 µg) with 100 µg denatured salmon sperm DNA was added to the cells in 0·5 ml 0·1 M lithium acetate, 40 % PEG 3350, 1 mM EDTA and 10 mM Tris, pH 7·5. The mix was incubated at 30 °C for 20 min, heat-shocked at 42 °C for 20 min and plated on SC-Ura plates for selection of transformants.
Plasmid constructs.
The YPS1 constructs were made by cloning the C terminus of YPS1 into pEpa1p(33-933) (Frieman & Cormack, 2002) whose expression is under the control of a galactose-inducible (GALS) promoter. The YPS1 fragment was amplified by PCR with primers 905 (5'-AACCGCTCGAGTAAGGCACCAGGCTATACAAACACTTG-3') and 616 (5'-GCGAATTCTTATCAGATGAATGCAAAAAGAAGAGAAATTAATG-3'), digested with XhoI and EcoRI and ligated into an appropriately digested vector. The resulting plasmid was named pBC570. Constructs ST861960
YPS1, ST761960
YPS1, ST633960
YPS1, ST583960
YPS1 and STFLO1
YPS1 were made by PCR amplification of the appropriate fragments and cloning into the XhoI site of pBC570. Construct ST861960
YPS1 was made with primers 1915 (5'-CCGGCTCGAGGCTATTTCCAAAAATGGTAAGGTGT-3') and 1912 (5'-CCGGCTCGAGTATAACAAGCTTAAACAATCCAGGT-3'). ST761960
YPS1 was made with primers 1915 (see above) and 1913 (5'-CCGGCTCGAGTCTTGATTATGACTGTTCGTTAGTC-3'). ST633960
YPS1 was made with primers 1915 (see above) and 498 (5'-AACCGCTCGAGTACCTCTATAAGCGTACCCTTC-3'), and ST583960
YPS1 was made with primers 1915 (see above) and 1914 (5'-CCGGCTCGAGTGTGCGCTCAACACTTCCTTCATCA-3'). STFLO1
YPS1 was made with primers 2395 (5'-ACGCTCTCGAGAGAAACTGTGATTGTTATC-3') and 1065 (5'-CTCGAGAACTTTGTTGTCTCTGTAGTAGAAATAGGGC-3'). The pHA-
CWP2 vector was made by amplification by PCR of a piece of CWP2 with primers 903 (5'-AACCGCTCGAGTGAATCCGCTGCCGCCATTTCTCAAAT-3') and 621 (5'-GCGAATTCTTATTATAACAACATAGCAGCAGCAGCTAGAGCACC-3'), which was then cloned into plasmid pBC214 (Frieman & Cormack, 2002) as an XhoI- and EcoRI-digested fragment. The same FLO1 fragment amplified to make STFLO1
YPS1 was cloned into the pHA-
CWP2 vector as an XhoI-digested fragment to make STFLO1
CWP2. ST633960
CWP2 was made by amplification of a region of EPA1 with the primers 1915 (see above) and 498 (see above) and cloned into the Xho1 site in pHA-
CWP2 plasmid.
FACS analysis.
Constructs were transformed into the yeast strain BY4742 selecting for growth in the absence of uracil. For analysis, strains were grown overnight in media lacking uracil, supplemented with 2 % raffinose. These cells were induced for expression of the fusion proteins by addition of 2 % (w/v) galactose and subsequent culture for 2 h. No agglutination of yeast was observed for any of the constructs analysed. The yeast cells were collected by centrifugation and labelled with mouse anti-haemagglutinin (HA) antibody (50 µg ml1) (Sigma-Aldrich) for 30 min at 4 °C on a rotating wheel. The yeast cells were then washed three times with PBS and labelled with secondary FITC-conjugated goat anti-mouse antibody (50 µg ml1) (Santa Cruz Biotechnology). Yeast cells were again washed three times in PBS and resuspended in 1 ml PBS for FACS analysis.
Protein extraction.
Cells were grown overnight in SC-Ura medium plus 2 % raffinose. For galactose induction, 2 % galactose (final concn) was added to the culture and induced for 2 h. Cells were collected by centrifugation and resuspended in 1 ml 50 mM Tris/HCl, pH 7·5. Then 0·5 ml glass beads were added and placed in a Bead Beater (Biospec) for 2 min. Broken cells were washed from the glass beads and the extract was pelleted at 15 000 g for 10 min to spin down all cell wall and membrane material. After removal of supernatant the pellet was then boiled for 10 min in 50 mM Tris/HCl, pH 7·5, containing 2 % SDS. The SDS-extractable material was saved and the remaining pellet was boiled again in 50 mM Tris/HCl, pH 7·5, containing 2 % SDS. The cell debris was collected by centrifugation and washed two times in 1 ml H2O. The cell debris was again pelleted and the pellet was resuspended in 100 µl 67 mM potassium phosphate. This is the washed cell wall material. Proteins were released from the washed cell wall material by treatment with 1,3-
-glucanase (Quantazyme; Qbiogene) as follows. A sample of the washed cell wall material (20 µl) was added to 75 µl phosphate buffer with 2 units 1,3-
-glucanase and 0·1 µl
-mercaptoethanol. The reaction was incubated at 30 °C for 2 h. The reaction was then pelleted for 5 min at 15 000 g and the supernatant removed for analysis. For all Westerns comparing cell wall and membrane fractions, we loaded amounts of 1,3-
-glucanase releasable material and SDS-extractable material corresponding to the same number of yeast cells.
Western analysis.
Proteins from cell extracts were separated by SDS-PAGE on 38 % gradient gels (Invitrogen). After transfer to PVDF membrane (Amersham Pharmacia Biotech), membranes were incubated overnight with mouse monoclonal IgG horseradish peroxidase-conjugated anti-HA antibody (Santa Cruz) at a concentration of 1 : 10 000 in 5 % non-fat milk/TBS. The blots were washed two times in 2 % Tween/TBS for 30 min and antibody was detected using ECL-Plus (Amersham Pharmacia Biotech).
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RESULTS |
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To distinguish between these two possibilities, we carried out Western analysis on membrane and cell wall fractions from cells expressing the various constructs in Fig. 2. Cells expressing each construct were grown in parallel and fractionated into cell wall and membrane fractions. Both fractions were separated by SDS-PAGE gel electrophoresis and analysed by Western blotting. The cell wall fractions contain very high molecular mass species that are both glycosylated and modified by covalent attachment to glucan. In the membrane fractions, we have previously shown that the fast-migrating species (Fig. 3
, lanes 6, 8, 10, 12 and 14) are endoplasmic reticulum intermediates. The slower species found in the membrane fractions have been further glycosylated in the Golgi (Frieman & Cormack, 2003
). As shown in Fig. 3
, there is very little if any material found in the cell wall lanes for
YPS1 and ST861960
YPS1, even though the protein is abundantly expressed in the membrane fraction. A very small fraction of protein can be seen in the cell wall lane for ST761960
YPS1, although most of the protein is in the membrane fraction. Constructs ST633960
YPS1 and ST583960
YPS1, which contain 328 and 378 aa of Ser/Thr, respectively, are primarily localized to the cell wall. As a control, we analysed constructs
CWP2 and ST633960
CWP2, both of which contain a GPI anchor signal of the GPI-CWP class; as expected, both are primarily localized to the cell wall (Fig. 3
). This effect was not specific to the Ser/Thr region derived from EPA1 since the Ser/Thr region derived from the FLO1 gene of S. cerevisiae was also able to redirect the GPI-PMP
YPS1 to the cell wall (Fig. 2b
). These experiments demonstrate that the addition of 200300 aa rich in serine and threonine residues can redirect a model GPI-PMP to the cell wall.
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DISCUSSION |
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GPI proteins can be conveniently divided into two groups based on a sequence feature first noticed by Klis (Caro et al., 1997) proteins with a dibasic motif in the amino acids immediately upstream of the
site are typically destined for the plasma membrane while GPI proteins lacking that signal are destined for the cell wall. The validity of this rule has been largely borne out by mutagenesis studies on model GPI proteins which clearly show that the distribution between cell wall and plasma membrane can be dramatically altered by point mutations in the region immediately upstream of the
site (the
-proximal site), including introduction or elimination of a dibasic motif. That said, for certain natural proteins, the rule is an imperfect predictor of ultimate localization. For example, even though Gas1p has traditionally been annotated as a GPI-PMP, it can clearly be found at the cell wall (De Sampaio et al., 1999
). One complication in using the
site sequence to predict cell wall versus plasma membrane localization is that there can be multiple potential
sites in one protein. Without protein sequence data, it is not possible to predict which of the multiple
sites is used. It is possible, therefore, that GPI protein species in cell wall or membrane fractions may in fact have different sites of GPI anchor attachment, and therefore different
-proximal signals.
In addition to the above considerations, localization of natural GPI proteins to the cell wall or membrane can clearly be affected by other features of the protein. In this paper, we show that for a constant GPI anchor addition sequence, the distribution between membrane and cell wall can be strongly affected by the sequence of the protein attached to the GPI anchor. For minimal GPI constructs (consisting essentially of an epitope tag and the GPI signal sequence), our previous work, as well as work from other labs (Frieman & Cormack, 2003; Hamada et al., 1998
, 1999
), found that localization to the plasma membrane or the cell wall is determined by the
-proximal signal and largely by the presence or absence of a dibasic motif. For larger chimeric proteins in which the GPI anchor is fused to the N-terminal domain of Epa1p (aa 1440), localization is again largely determined by the ·
site-proximal signal, since a fusion to the YPS1 anchor sequence is largely membrane-localized while a fusion to the CWP2 anchor sequence is largely cell-wall-localized. However, full-length Epa1p or chimeric fusions that contain approximately 300 aa of the Ser/Thr region of Epa1p (or Flo1p) are localized to the cell wall independent of the GPI-proximal signal, since fusions to both the YPS1 and CWP2 GPI anchor signals are cell-wall-localized. Thus, in addition to the ·
site proximal signals, the ultimate distribution of GPI proteins between cell wall and membrane is affected by the sequence of the protein (which we term the cargo) attached to the GPI anchor. In the case of regions derived from the Ser/Thr regions of Epa1p or Flo1p, this cargo signal can override the
-proximal site signal. The ability of the cargo to override the
-proximal site signal is not wholly determined by size, since the N-terminal domain of Epa1p (aa 1440) which does not override the
-proximal site signal (Fig. 1b
and Frieman & Cormack, 2003
) is larger than the Ser/Thr region (approx. 300 aa) which does override the
-proximal site signal. We have shown only that the Ser/Thr regions of Epa1p and Flo1p are sufficient to redirect a GPI-PMP to the cell wall. We think it is unlikely that a Ser/Thr region is the only domain that can override a GPI-PMP
-proximal site signal. Indeed, De Sampaio et al. (1999)
showed that Gas1p and fusions of Gas1p to
-galactosidase are both primarily cell-wall-localized. Neither Gas1p nor
-galactosidase is particularly rich in serine and threonine residues.
We tested whether mannosylation of the Ser/Thr regions of Epa1p or Flo1p by any single PMT-encoded mannosyl transferase was required for its ability to redirect a GPI-PMP to the cell wall. We found that only deletion of PMT2 had any effect at all. However, while deletion of PMT2 did result in a modest decrease in the total amount of epitope-tagged GPI protein as well as some decrease in the ratio of tagged protein in the cell wall versus the membrane fractions (Fig. 5), this effect was really significant only for GPI proteins containing the Epa1p Ser/Thr region; by contrast, deletion of PMT2 had much less effect on surface expression for constructs containing the Ser/Thr region from Flo1p or Flo9p (Table 1
). Even more importantly, loss of PMT2 affected trafficking of constructs containing the Ser/Thr region of Epa1p regardless of whether the GPI anchor was derived from YPS1 or CWP2. Thus, while PMT2 affects trafficking of constructs containing the Epa1p Ser/Thr region, we have no evidence that PMT2 plays a role in the ability of Ser/Thr regions to override the GPI-PMP
-proximal signal. An important caveat of our experiments with the mannosylation mutants is that there is only a partial loss of mannosylation in any of the pmt backgrounds as measured by change in mobility on SDS-PAGE gels (data not shown). This is not surprising, given the overlapping functions of mannosyl transferases, but it remains possible that in strains deleted for multiple PMT genes, for example, there would be an effect on the ability of the Ser/Thr-rich regions to override a GPI-PMP
-proximal signal.
The reduction in overall protein levels seen in the pmt2 background for the chimeras containing the Epa1p-derived Ser/Thr region (Fig. 5) could be due to reduced stability of the proteins in the pmt2 background, perhaps as a result of degradation of undermannosylated protein. Indeed, we could demonstrate a small reduction in the molecular mass of the chimeric Epa1-derived proteins in a pmt2 background, consistent with a role for PMT2 in mannosylating the Epa1p Ser/Thr domain (Fig. 5
). Interestingly, we found no equivalent effect of PMT2 on the Ser/Thr region derived from Flo1p or Flo9p, perhaps suggesting some specificity of PMT2 for the Epa1p Ser/Thr domain. Mannosylation of the Mid2p cell wall integrity sensor which has an extracellular domain rich in serine and threonine residues also specifically requires PMT2 (Philip & Levin, 2001
). The specific requirement for PMT2 in mannosylation of Mid2p or of the Epa1p Ser/Thr region is not understood.
We have shown that multiple, separable signals can be integrated to determine the ultimate localization of GPI proteins to the cell wall or plasma membrane. We found that a long Ser/Thr region could override the -proximal GPI signal of a predicted GPI-PMP to localize it to the cell wall. This raises the question as to the fate of actual GPI proteins in the cell, since GPI proteins apparently can contain at least two signals that in theory could direct different localizations. To begin to address this question, we examined the list of predicted GPI proteins in S. cerevisiae and found that the overall Ser/Thr content is low for predicted GPI-PMPs and high for the majority of predicted GPI-CWPs. It may be that this correlation simply reflects a functional role of Ser/Thr residues in GPI-CWPs rather than GPI-PMPs. It also suggests, however, that for the majority of GPI proteins in S. cerevisiae, the two signals we describe in this paper (the
-proximal signal and Ser/Thr content) might act in concert rather than antagonistically. While at least some of the cis-requirements directing localization of GPI proteins to the plasma membrane or cell wall are now understood, it remains to be determined how the cell actually interprets those sequence signals and sorts the two classes of GPI proteins.
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ACKNOWLEDGEMENTS |
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Received 18 June 2004;
accepted 14 July 2004.
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