Also, hybrid Ath1 truncates fused at their C-terminus with the yeast internal invertase revealed that a 131 amino acid N-terminal fragment of Ath1was sufficient to target the fusion prote
Trang 1targeted at the cell surface for its physiological function Susu He1,2,3, Kerstin Bystricky4,5, Sebastien Leon6, Jean M Franc¸ois1,2,3and Jean L Parrou1,2,3
1 University of Toulouse, INSA, UPS, INP & INRA, France
2 INRA-UMR 792 Inge´nierie des Syste`mes Biologiques et proce´de´s, Toulouse, France
3 CNRS-UMR 5504, Toulouse, France
4 Laboratoire de Biologie Mole´culaire Eucaryote, University of Toulouse, France
5 CNRS-UMR5099, Toulouse, France
6 Institut Jacques Monod, UMR7592 CNRS ⁄ Universite´ Paris Diderot, France
Introduction
Trehalose [alpha-d-glucopyranosyl (1fi 1)
alpha-d-gluocopyranoside] is a nonreducing disaccharide found
in many organisms including yeasts, fungi, bacteria,
plants and insects Trehalose is one of the major storage
carbohydrates in the yeast Saccharomyces cerevisiae,
accounting for > 25% of cell dry mass depending on
the growth conditions and the life-cycle stage of the yeast
[1–3] The accumulation of intracellular trehalose has
two potential functions First, it constitutes an
endoge-nous storage of carbon and energy during spore
germi-nation and in resting cells Second, trehalose acts as a
stabilizer of cellular membranes and proteins [4–6]
In S cerevisiae, trehalose is hydrolyzed to glucose
by the action of two types of trehalase: ‘neutral treha-lases’ encoded by NTH1 and NTH2 [3,7], which are optimally active at pH 7, and ‘acid trehalases’ encoded
by ATH1, which show optimal activity at pH 4.5 [8] Although fungal acid trehalases, including those of the yeast Candida albicans [9] and Kluyveromyces lactis [10], have been reported to be localized at the cell sur-face, the localization of the S cerevisiae acid trehalase remains a matter of controversy In 1982, Wiemken and co-workers [11] first identified this protein in a vacuole-enriched fraction obtained by density gradient
Keywords
acid trehalase; cell surface; fluorescence
microscopy; Saccharomyces cerevisiae;
secretion
Correspondence
J M Franc¸ois, University of Toulouse,
INSA, UPS, INP & INRA, 135, Avenue de
Rangeuil, F-31077, Toulouse, France
Fax: +33 5 6155 9400
Tel: +33 5 6155 9492
E-mail: fran_jm@insa-toulouse.fr
(Received 6 May 2009, revised 9 June
2009, accepted 21 July 2009)
doi:10.1111/j.1742-4658.2009.07227.x
Previous studies in the yeast Saccharomyces cerevisiae have proposed a vac-uolar localization for Ath1, which is difficult to reconcile with its ability to hydrolyze exogenous trehalose We used fluorescent microscopy to show that the red fluorescent protein mCherry fused to the C-terminus of Ath1, although mostly localized in the vacuole, was also targeted to the cell sur-face Also, hybrid Ath1 truncates fused at their C-terminus with the yeast internal invertase revealed that a 131 amino acid N-terminal fragment of Ath1was sufficient to target the fusion protein to the cell surface, enabling growth of the suc2D mutant on sucrose The unique transmembrane domain appeared to be indispensable for the production of a functional Ath1, and its removal abrogated invertase secretion and growth on sucrose Finally, the physiological significance of the cell-surface localization of Ath1 was established by showing that fusion of the signal peptide of invertase to N-terminal truncated Ath1 allowed the ath1D mutant to grow on trehalose, whereas the signal sequence of the vacuolar-targeted Pep4 constrained Ath1
in the vacuole and prevented growth of this mutant on trehalose Use of trafficking mutants that impaired Ath1 delivery to the vacuole abrogated neither its activity nor its growth on exogenous trehalose
Abbreviations
EndoH, endoglycosidase H; GH, glycolsyl hydrolase; GP, green fluorescent protein; MVB, multivesicular body; TM, transmembrane.
Trang 2centrifugation of a yeast protoplast preparation The
vacuolar localization of acid trehalase was very
recently supported by in vivo imaging analyses using
green fluorescent protein (GFP)–Ath1 fusion
con-structs under the strong and constitutive TPI1
pro-moter [12] Furthermore, Huang et al used various
trafficking mutants to show that this acid trehalase
reaches its vacuolar destination via the multivesicular
body (MVB) pathway However, this localization
con-trasts with the fact that this enzyme allows yeast to
grow on exogenous trehalose [13] and with measurable
Ath1 activity at the cell surface [14]
The purpose of this study was to revisit this
contro-versy regarding the localization of Ath1 in light of its
biological function, combining cell biology and
bio-chemical approaches To this end, we investigated the
localization of Ath1 using strains expressing the red
fluorescent protein mCherry fused to the C-terminus of
Ath1 Integration of this construct at the ATH1 locus
had the advantage of expressing the protein at levels
comparable with those in wild-type cells, because it is
reported that overexpression may cause the
mislocal-ization of proteins into the vacuoles [15], and also to
investigate the fusion protein under physiological
con-ditions The domain responsible for targeting Ath1 at
the cell surface and the role of the single
transmem-brane (TM) domain at the N-terminus of this protein
were investigated The functional localization of Ath1
was further assessed by constructing various Ath1
hybrid proteins bearing different targeting signal
pep-tides Together, our results demonstrated that the
localization of Ath1 at the cell periphery is required
for growth on trehalose, whereas the vacuolar
localiza-tion of this protein is not compatible with growth on
this carbon source
Results
Ath1 is localized at the cell periphery
In a previous report, the localization of S cerevisiae
Ath1 was visualized using a pGFPATH1 construct
that expressed a GFP fused to the N-terminus of Ath1
under the strong TPI1 promoter [12] We obtained a
comparable result with a GFP–Ath1 construct that
was expressed under the control of the
methionine-repressible MET25 promoter in a glucose medium
lacking methionine (Fig 1A) However, western
blot-ting using a GFP antibody on extracts from cells
expressing GFP–Ath1 revealed a major band migrating
at a position corresponding to 30 kDa, instead of
bands migrating at > 150 kDa (Fig 1B) Fluorescence
in the vacuole may therefore be caused by free GFP
which accumulated in this organelle because it has been reported that targeting of GFP-fusion proteins to the vacuolar lumen leads to their degradation by vacu-olar proteases However, this degradation process is usually delayed, leading to the transient accumulation
of GFP-containing proteolytic fragments of 30 kDa, and a sustained luminal vacuolar fluorescence [16] Note that a similar result was reported by Huang et al [12], although they were also able to detect a band corresponding to the native GFP–Ath1
This proteolytic problem, coupled with the fact that overexpression under a strong promoter has been reported to mislocalize some proteins into vacuoles [15], prompted us to re-examine the localization of Ath1 by fusing of GFP to its C-terminus, and express-ing the correspondexpress-ing ATH1–GFP fusion gene under the native promoter after integration at ATH1 locus Under this condition, we were able to observe a green signal at the cell periphery, although most of the signal was still localized in the lumen of the vacuole (Fig 1C) Similar results were obtained using the red fluorescent protein mCherry, which was also integrated
at the ATH1 chromosomal locus, as well as with the tag fused at the N-terminus of Ath1 (data not shown)
As for Ath1–GFP or GPF–Ath1 (see above), the Ath1–mCherry fusion protein was fully functional as indicated by the growth of this recombinant strain on trehalose and by enzymatic measurement (see below) Under live cell fluorescence microscopy, we observed a strong signal in the vacuolar compartment together with a clearly discernable signal at the cell periphery (Fig 1D) These results indicated that Ath1 may have two localizations, one in the vacuole, in agreement with previous studies [11,12], and another at the cell periphery, in accordance with its ability to hydrolyze exogenous trehalose [14] We then verified the Ath1– mCherry fusion protein by western blot This analysis made on extracts from yeast cells expressing the chime-ric protein revealed a band at a size > 200 kDa with the rabbit anti-DsRed sera (Fig 1E) Because this sig-nal disappeared upon endoglycosidase H (EndoH) treatment, the glycosylation that was reported for this protein [7] may explain this migration property at an apparent size much higher than expected However, the expected band at a size of 164 kDa (Ath1 + mCherry) was barely detected upon EndoH treatment, and instead, a relatively strong band migrat-ing at around 65 kDa could be identified (Fig 1E)
As a second, independent way to support the locali-zation of Ath1 at the cell periphery, we used the invertase secretion system Invertase is a secreted pro-tein with a classical signal peptide at its N-terminus (amino acids 1–19) for secretion at the cell periphery
Trang 3Deletion of this signal peptide (suc2ic allele) prevents
secretion and results in the accumulation of the
trun-cated form of the enzyme in the cell, impairing the
ability of S cerevisiae to grow on sucrose or raffinose
as the sole carbon source We generated an inframe
fusion of full-length ATH1 and suc2ic (pSC1–ATH1),
leading to the chimeric Ath1–Suc2 protein expressed
under the ATH1 promoter As shown in Fig 2, suc2D
mutant expressing this gene construct recovered
growth on sucrose, like the positive control expressing the full-length secreted invertase under its own pro-moter (pLC1), whereas suc2D mutant transformed with pSC1 lacking of signal peptide grew very poorly on sucrose, probably using amino acids present in the medium (Fig 2B) Consistent with this, these cells also recovered invertase activity in both crude extract and intact cells (Fig 3), albeit five times lower than that measured in suc2D mutant transformed with pLC1
Fig 1 Cellular localization of Ath1 Yeast cells in the exponential phase (D 600 1.0) expressing fusions of Ath1 to GFP or mCherry were collected for live cell micros-copy (A,C,D) and western blotting (B,E) Bar length = 2 lm (A,B) BY4742 ath1D trans-formed with pGFP–ATH1 under the MET25 promoter in YN glucose without methionine (A) (left) fluorescence, (right) DIC (B) Immu-noblot with anti-GFP of crude extract before ( )) and after (+) N-deglycosylation by EndoH; (C) BY4741 bearing ATH1–GFP integrated at the ATH1 locus in YN trehalose (D,E) BY4741 bearing ATH1– mCherry integrated at the ATH1 locus in YN trehalose (D) Microscopy and (E) immuno-blot with the DsRed polyclonal antibody of crude extract before ( )) and after (+) N-deglycosylation by EndoH M, Molecular mass marker; arrowhead, expected full-length fusion protein at 150 kDa; asterisk, degradation product.
Fig 2 Complementation of the S cerevisiae SEY6210 strain (suc2D mutant) with different Ath1–invertase chimera (A) Schematic representa-tion of the different gene fusion constructs pSC1, negative control (invertase without signal peptide); pLC1, positive control (full-length invertase); for the remaining constructs, the Suc2 signal peptide has been replaced by full-length ATH1 sequence (pSC1–ATH1) or N-terminal sequence variants of ATH1 with decreasing size; (B) growth on YP medium with sucrose (complementation test) or glucose (control) for 5 days.
Trang 4Replacing the ATH1 promoter in pSC1–ATH1 with
the stronger SUC2 promoter resulted in an invertase
activity similar to that in pLC1 (data not shown) We
noticed that the invertase activity in a crude extract of
cells transformed with pLC1 was lower than that in
intact cells This may be caused by incomplete lysis of
the cells or partial denaturation of proteins during
extraction and vortexing with glass beads
In addition to the cell biology data, we also
revali-dated our enzymatic assay of acid trehalase Our
cur-rent method is based on the measurement of the
activity in intact cells according to the procedure
employed to measure secreted invertase [17], in which
NaF is added to the incubation mixture to block
glu-cose uptake We verified that the use of NaF did not
cause any enzymatic artifact, for example, cell lysis or
the release of intracellular glucose First, incubation of
intact cells from an exponential culture grown on
glucose that do not express acid trehalase because of
glucose repression [18] in a reaction mixture optimal
for neutral trehalase activity and containing NaF did
not lead to any glucose production from trehalose
(data not shown) This excluded the possibility of cell
leakage and the release of proteins or intracellular
glu-cose under NaF treatment Further validation of our
assay was the successful measurement of acid trehalase
activity on intact cells from a mutant completely
defec-tive for glucose uptake (hxt1-17D strain) [19] cultivated
on glycerol and ethanol as the carbon source, which
allowed ATH1 expression, even in the absence of NaF
(data not shown) These elements demonstrated that
the glucose measured in intact cells resulted from
cleavage of the disaccharide by an acid trehalase local-ized at the cell surface
Searching for the minimal domain of Ath1 for invertase secretion
Full-length Ath1–invertase fusion protein was targeted
at the cell surface, suggesting the existence of a secre-tion sequence in Ath1 As shown in Fig 4, domain prediction using the smart program [20,21] did not reveal any classical signal peptide for secretion at the N-terminus of Ath1 This in silico analysis only revealed a short 23 amino acid TM domain near the N-terminus, followed by three ‘glycosyl hydrolase’ (GH) domains (amino acids 132–415, 474–845 and 849–904) that together may constitute the catalytic domain of Ath1 [22] To map the minimal domain of Ath1 that allows the secretion of this protein, various DNA fragments of ATH1 were fused inframe with the suc2ic allele (Fig 2A) A series of plasmids, namely pSC1–N that carried a fusion to the first 131 N-termi-nal amino acids of Ath1, pSC1–TM bearing a fusion
to the first 69 amino acids of Ath1, which includes the
TM domain, and pSC1–tm that only bears the first 46 amino acids of Ath1 excluding the TM domain, were introduced into the suc2D mutant SEY6210 Transfor-mants were tested for growth recovery on sucrose (Fig 2B) and for invertase activity (Fig 3) As shown
in Fig 2B, suc2D mutant cells transformed with pSC1–
N or pSC1–TM were able to grow on YP sucrose as readily as pSC1–ATH1, whereas cells transformed with pSC1–tm poorly grew on sucrose, as did cells bearing the negative control pSC1
Invertase activity was measured in intact cells and crude extracts from suc2D mutant transformed with these various constructs, compared with growth effi-ciency on sucrose (Figs 2 and 3) Cells transformed with pSC1–N showed an activity nearly twofold higher than that in cells expressing a fusion to the full-length Ath1 (pSC1–ATH1) One explanation might be that the full size Ath1 fused to internal invertase somehow
Fig 3 Invertase activity in the S cerevisiae SEY6210 strain (suc2D
mutant) transformed with the various Ath1–invertase constructs.
The constructs are those shown in Fig 2 Transformed cells were
cultivated in YP sucrose medium until the stationary phase before
measurement of invertase activity on intact cells and in crude
extract as described in Experimental procedures Histograms show
the results of two independent experiments (mean ± SD).
Fig 4 Ath1 predicted functional domain using the SMART program Theoretical glycosylation sites (yellow triangles); N-terminal trans-membrane segment (TM); N-term (GH_65N), central (GH_65m) and C-term (GH_65C) domains from the CAZy glycoside hydrolase family 65 The latter three domains likely constitute the catalytic core of trehalase.
Trang 5impairs folding of the invertase domain and⁄ or the
catalytic efficiency on its substrate Despite this
differ-ence, as for pSC1–ATH1, the activity in intact cells
was comparable with that in cell extract, and both
transformed cells showed similar qualitative growth on
sucrose The activity measured in intact cells
express-ing pSC1–TM was four times lower than that in the
crude extract, and two to four times lower than in
intact cells transformed with pSC1–ATH1 and
pSC1–N Bearing in mind this low activity, pSC1–TM
transformed cells were found to grow slightly more
slowly on sucrose than cells transformed with pSC1–
ATH1 Further reduction at the N-terminus (i.e with
pSC1–tm) resulted in residual invertase activity in the
crude extract, together with an inability to grow on
sucrose Taken together, these results showed that a
minimal fragment of 69 amino acids encompassing the
unique TM domain of Ath1 was needed to promote
correct expression of the internal invertase, but was
not sufficient for efficient protein secretion, which was
achieved with a 131 amino acid N-terminus of Ath1
Removal of the N-terminus of Ath1 caused a
strict vacuolar localization
Because the 131 amino acid N-terminus of Ath1
appeared to be sufficient for invertase secretion, we
further investigated the targeting properties of this
fragment by using a mCherry fusion that was
expressed under the control of the ATH1 promoter
(pN–mCherry) Figure 5A shows a fluorescent signal
at the cell periphery and a stronger signal in the
vacu-ole, similar to that observed using full-length Ath1
fused to mCherry (compare Figs 5A and 1D) This
result confirmed that the N-terminal part of Ath1 was
sufficient to target the recipient protein to these two
cellular compartments
Reciprocally, we analyzed the consequences of
delet-ing the first 100 codons of the ATH1 sequence
(path1DN) on red protein localization When expressed
in a wild-type strain grown on trehalose, the Ath1DN–
mCherry fusion protein led to a fluorescent signal
exclusively in the vacuole (Fig 5B) No discernable
signal could be detected at the cell periphery, even
after 10-fold longer exposure times From this result,
we first verified that a BYath1D mutant transformed
with the centromeric plasmid pATH1 carrying the
wild-type ATH1 gene recovered wild-type
characteris-tics, i.e growth on trehalose as the sole carbon source
(not shown), and acid trehalase activity in both intact
cells and cell crude extracts (Fig 6) However, when
this ath1D mutant was transformed by path1DN it was
not able to grow on trehalose (data not shown) and
had no Ath1 activity (Fig 6) From these data, we were able to confirm that the 131 amino acid N-termi-nal fragment contains important information for cell-surface targeting, and we suggest that there may
be vacuolar targeting determinants in the catalytic domain, as in the case of acid phosphatase [23]
Fig 5 The N-terminal domain of Ath1 is needed for the localization
of mCherry at the cell periphery Live cell microscopy of exponen-tially growing cells in YN trehalose medium of the BY4741 strain transformed with pN–mCherry (A) or with path1DN–mCherry (B).
Fig 6 Acid trehalase activity of ath1D mutant cells expressing vari-ous ATH1 constructs The BY4741 ath1D mutant strain transformed with gene constructs expressing different Ath1 variants was culti-vated in YN glucose to late exponential phase (D600 8) with plas-mid selection The cells were then collected and transferred to YPD medium until the stationary phase (D 600 20) to allow ATH1 derepression Acid trehalase activity was measured in intact cells and crude extracts as described in Experimental Procedures Histograms show the results of two independent experiments (mean ± SD).
Trang 6Substitution of the N-terminus of Ath1 by the
invertase signal peptide restored acid trehalase
activity and growth on trehalose
The exclusive, strong vacuolar signal observed in the
absence of the 100 amino acid N-terminus of Ath1,
together with the subsequent loss of catalytically active
trehalase (Ath1DN variant), suggested that the
vacuo-lar fraction consisted mainly of inactive Ath1 We
therefore asked whether targeting of Ath1 to the cell
periphery could restore trehalase activity We made
use of the invertase secretion property by fusing the
signal peptide of this protein to the N-terminus of the
Ath1DN variant Fig 7A) When transformed in ath1D
mutant cells, the resulting plasmid pSPSUC2–
ATH1DN did allow recovery of the growth ability on
trehalose and the acid trehalase activity in both cell
crude extract and intact cells (Fig 6) Moreover, the
ath1D mutant strain bearing this plasmid grew about
two times faster than wild-type BY4741 strain on
syn-thetic trehalose medium (l = 0.10 versus 0.047;
Fig 8) Localization of this hybrid protein was verified
by C-terminal fusion to mCherry Setting our exposure
time as in Fig 1, we found that the intensity of the
fluorescent signal at the cell periphery was significantly
higher than that of the full-length Ath1–mCherry
pro-tein (compare Figs 7B and 1D) However, the bulk of
the fluorescent signal still resided in the vacuolar
com-partment, which substantiated the idea that the
cata-lytic domain of Ath1 contains some targeting signal
for the vacuole Using western blot analysis, we found
a 65kDa proteolytic fragment that was already
obtained with the Ath1–mCherry fusion protein (Fig 1C), but also a clearly detectable band corre-sponding to the SPSuc2–Ath1DN–mCherry chimeric protein after EndoH treatment (173 kDa, Fig 7C), indicating better stability for this construct than for native Ath1 Overall, these results suggest that secre-tion of Ath1 at the cell periphery is associated with the stabilization and physiological function of this protein
Constraining Ath1 to the vacuole impaired growth on trehalose
Although Ath1 can be targeted to the cell periphery, the vacuolar localization appeared to be the major des-tination for this protein, as illustrated by the strong vacuolar signal obtained using fluorescence micros-copy To check the possible function of the vacuolar pool of acid trehalase for growth on trehalose, we sought a strategy to constrain all Ath1 in this intracel-lular compartment To this end, we fused the signal peptide of the vacuolar protein Pep4 [24] to the N-ter-minus of the truncated Ath1DN variant Very interest-ingly, when transformed in ath1D mutant cells, the plasmid pSPPEP4–ATH1DN did not allow recovery of the growth on trehalose (Fig 8), although the cells did exhibit acid trehalase activity in the crude extract, which accounted for 50% of the activity measured
in cells expressing SPSuc2–Ath1DN (data not shown)
As shown in Fig 7D, microscopy analysis confirmed that the SPPep4–Ath1DN–mCherry chimeric protein was exclusively targeted to the vacuole when expressed
in the wild-type strain This strongly indicated that the
Fig 7 Cellular localization of the Ath1
cata-lytic core fused to different signal peptides.
(Panel I, A) Schematic representation of
SUC2, PEP4 and ATH1 nucleotide
sequences with emphasis on the 5¢-end
containing targeting information Nucleotides
1-111 and 1-267 of SUC2 and PEP4,
respec-tively, replaced nucleotides 1-300 of ATH1
giving the SPSUC2–Ath1DN and SPPEP4–
Ath1DN chimeras (Panel II) BY4741 cells
were transformed with pSPSUC2–ath1DN–
mCherry and cultivated in YN trehalose
medium Exponential growing cells were
collected for live cell microscopy (B), and
immunoblot on crude extract before ( )) and
after (+) deglycosylation with EndoH using
DsRed polyclonal antibody (C) (D) BY4741
cells transformed with pSPPEP4–ath1DN–
mCherry were collected for live cell
micros-copy M, molecular mass markers;
arrow-head, expected full-length fusion protein;
asterisk, degradation product.
Trang 7vacuolar pool of acid trehalase has no role in trehalose
assimilation for cell growth
As a complementary approach, we used mutants of
genes involved in the vacuolar sorting pathway, like
VPS4 which encodes a protein implicated in the
deliv-ery of proteins from the prevacuolar compartment to
the vacuole [25] As shown in Fig 9A, the intracellular
red fluorescent signal derived from Ath1–mCherry was
totally mislocalized in a vps4D mutant, being
com-pletely excluded from the lumen of vacuole However,
the fluorescent signal at the cell periphery was still
visi-ble in this vps4D mutant and the relative Ath1 activity
between intact cells and crude extract was identical to that of wild-type cells (Fig 9B) The presence of the Ath1–mCherry fusion protein was also monitored in this mutant using the rabbit anti-DsRed sera In untreated extract, a band migrating at 200 kDa was relatively comparable in this mutant and the wild-type (data in Figs 9C and 1C can be compared because sim-ilar amount of protein were loaded) After EndoH treatment, the expected 164 kDa band was visible, whereas the abundance of the 65 kDa band was drasti-cally reduced compared with that in Fig 1C, indicat-ing significantly decreased proteolysis of this protein when preventing vacuolar targeting Together, these results confirmed that trehalase in the vacuole is likely prompted to partial degradation and is not required for cell growth on trehalose
The TM domain is indispensable for Ath1 function
Previous studies have indicated that the short TM domain located at the N-terminus of Ath1 contained sufficient signaling information to deliver Ath1 to the vacuole via the MVB pathway [12] As already observed when studying invertase fusions, the require-ment for a minimal N-terminal fragrequire-ment encompassing the TM domain indicated the importance of this domain in protein expression and secretion (see the minimal construct pSC1–tm in Figs 2 and 3) We confirmed this by studying pSC1–ath1DTM and pSC1–NDTM, in which the TM domain was specifi-cally deleted in the full-length ATH1–SUC2 gene
Fig 8 Growth complementation of the ath1D mutant strain on
tre-halose After a preculture on a selective YN glucose medium, the
BY4741 ath1D mutant strain expressing the catalytic domain of
Ath1 (amino acids 301 to 1211) fused to signal sequence of Suc2
and Pep4, respectively, were transferred in YN trehalose medium
to evaluate growth complementation BY4741, positive control;
ath1D, negative control.
Fig 9 Localization and activity of Ath1 in vps4D mutant Cells cultivated in YN treha-lose medium to the exponential phase were collected for live cell microscopy (A), and to test the activity of acid trehalase in intact cells or cell crude extracts (B), as described
in Experimental Procedures Histograms show the results of two independent experi-ments (mean ± SD) (C) Crude extract from vps4D cells expressing Ath1–mCherry immunoblotted with the DsRed polyclonal antibody, before ( )) and after (+) deglycosylation with EndoH M, molecular mass markers Bar = 2 lm.
Trang 8fusion and in its truncated variant, respectively When
transformed in suc2D mutant, these constructs did not
restore growth on sucrose or invertase activity
(Fig 10A) Similarly, when using BYath1D mutant as
a recipient strain for functional complementation by
various Ath1 variants, the plasmid path1DTM, which
expressed an Ath1 protein lacking the TM domain,
was not able to complement growth deficiency of this
mutant on trehalose or yield measurable Ath1 activity
in this strain (data not shown) Finally, when using the
pNDTM–mCherry plasmid that expressed a fusion of
the N-terminal fragment lacking the TM domain to
mCherry, the fluorescence was observed in cytoplasmic
patches distinct from the vacuole (Fig 10B)
The absence of Ath1 activity in crude extracts when
TM was deleted from the protein prompted us to
ver-ify whether removal of this short TM domain may
hamper expression of these constructs For this
pur-pose, Ath1 and its ath1DTM variant were tagged with
3HA at their N-terminus The ath1 mutant
trans-formed with pHA–ATH1 expressing the Ath1–HA
fusion protein recovered growth on trehalose, although
the chimeric protein could not be detected by western
blotting, probably because of its very low expression
level We therefore replaced the ATH1 promoter with
the strong, inducible GAL1 promoter, leading to very
high Ath1 activity in cells transformed with pPGAL1–
HA–ATH1 (data not shown) By contrast, no activity
was measured in cells transformed with pPGAL1–HA– ath1DTM, although the gene construct was expressed (data not shown) These results were confirmed by western blot analysis using anti-HA IgG, which revealed a band at 130 kDa (wild-type Ath1) after EndoH treatment of protein extracts from cells expressing pPGAL1–HA–ATH1; no band was detected when the TM domain was missing from the protein (Fig 10C) These results supported the idea that absence of the TM domain may lead to a deficiency in protein production, which likely occurred during the early steps of endoplasmic reticulum protein synthesis and⁄ or during folding
Discussion
Vacuolar Ath1 is also found at the cell surface Controversy concerning the localization of Ath1 has been raised in two recent papers In a previous study,
we suggested a localization for Ath1 at the cell surface based on enzymatic data because most Ath1 activity could be measured in intact cells [14], in a manner sim-ilar to that for the secreted invertase [17] However, Huang et al [12] provided several arguments for a strict vacuolar localization of Ath1, identifying the MVB pathway as the main transport route for sorting this protein into the vacuole In this paper, we used
Fig 10 Role of the single transmembrane (TM) domain in protein expression (A) Left, schematic view of chimera proteins Ath1DTM–invert-ase and NDTM–invertAth1DTM–invert-ase, respectively Right, complementation tests of Suc2D mutant by these two constructions on YP sucrose agar for
5 days, and invertase activity (IA) (B) BY4741 cells transformed with plasmid pNDTM–mCherry were cultivated in YN trehalose medium to the exponential phase and collected for live cell imaging (C) The HA–ATH1 or HA–ATH1DTM gene constructs expressing Ath1 with or without the TM sequence tagged with HA under the GAL1 promoter were transformed into ath1D mutant cultivated in YN galactose EndoH-treated crude extracts were immunoblotted with the anti-HA IgG Lane 1, wild-type Ath1 (negative control); lane 2, HA–Ath1; lane 3, HA–Ath1DTM M, molecular mass markers.
Trang 9two independent methodologies, fluorescence
micros-copy and gene fusion to invertase, which together
pro-vided evidence that Ath1 is also targeted to the cell
surface Using the GFP or the red fluorescent protein
mCherry fused to the C-terminus of Ath1, we clearly
observed a localization of Ath1 at the cell periphery,
although the bulk fluorescent signal was still seen in
the vacuole A possible reason for the failure of Huang
et al [12] to find Ath1 at the cell periphery may be
that these authors used a GFP–Ath1 construct that
was expressed from the strong constitutive TPI1
pro-moter, because we obtained similar results using GFP–
Ath1 expressed from another strong MET25 promoter
However, we examined the localization of Ath1 in cells
expressing either Ath1–GFP or Ath1–mCherry
culti-vated on trehalose, whereas Huang et al [12]
investi-gated this localization problem using exponentially
growing cells on glucose It can be proposed that the
correct localization of Ath1 is dependent on the
sub-strate (in this case, trehalose), as shown for the control
of Fur4 permease by uracile [26] When expressed
under its own promoter, as in our study, ATH1 is
repressed by glucose [18] and the localization of Ath1
can be examined only in the stationary phase Thus,
the use of a glucose medium to study the localization
of Ath1 can be cautioned because it is not
physiologi-cally relevant for this protein Further evidence for a
cell-surface localization of Ath1 was obtained by
show-ing that expression of the Ath1–Suc2 protein fusion
allowed recovering suc2D mutant to grow on sucrose,
indicating that the full-length Ath1 protein was able to
drive the yeast internal invertase to the cell surface
These cell biology data were further supported by the
revalidation of our enzymatic assay of acid trehalase
on intact cells, confirming that glucose measured in
NaF-treated intact cells results from the cleavage of
the disaccharide at the cell surface by an extracellular
‘acid trehalase’ pool [14]
The cell-surface localization accounts for growth
on trehalose
It is known that Ath1 hydrolyzes exogenous trehalose
to grow on this carbon source Based on an exclusive
vacuolar localization for this protein, two models have
been proposed [27] The first suggested that Ath1 is
transported to the plasma membrane where it binds to
trehalose located at the cell surface; both trehalose and
trehalase are then internalized by endocytosis into the
vacuole where hydrolysis takes place According to
the results of Huang et al [12], this model may be
discarded because transport of Ath1 via the MVB
pathway en route to the vacuole bypasses the plasma
membrane The second model considered that treha-lose alone is delivered to the vacuole by endocytosis, where it is hydrolyzed by the resident Ath1 However, this model requires the identification of a trehalose endocytosis process and this is difficult to reconcile with mono- and disaccharides entering the cell by sugar permeases [19], and yeast cells possessing a high-affinity trehalose transporter encoded by AGT1 [28] Instead, we provide arguments that support a more simple model [14], in which trehalose can be assimi-lated by either a Agt1–Nth1 pathway, implicating the uptake and intracellular hydrolysis by neutral treha-lase, or by direct hydrolysis of trehalose by the extra-cellular acid trehalase encoded by ATH1 into glucose, which is thereafter taken up by the cells These two pathways only function in a MAL-positive strain such
as the CEN.PK background because expression of AGT1 is MAL dependent Because the sequenced BY4741 strain is mal-negative, the assimilation of exogenous trehalose can rely only on the Ath1-depen-dent pathway [14] Moreover, this model is consistent with what has been shown for fungal and plant acid trehalases, which are all localized at the cell surface or cell wall [22,29,30] In addition to these data, other results support this model First, constraining acid tre-halase in the vacuole by replacing its 100 amino acid N-terminal fragment with the signal sequence of the vacuolar Pep4 [24], a protein known to be specifically targeted to the vacuole, prevented growth on trehalose Second, impairment of Ath1 delivery to the vacuole using vps4D mutants defective in the MVB pathway did not abrogate growth on trehalose or the activity of Ath1 on intact cells
Although Ath1 is present at the cell periphery, our data,together with those from Huang et al [12], showed an apparent large accumulation of this protein
in the vacuole, as monitored by the fluorescence inten-sity from GFP- or mCherry-tagged protein However, this result contrasted with enzymatic data showing that Ath1 activity measured in crude extract was only 20–40% higher than that measured in intact cells One explanation for this discrepancy can be found from western blot analysis in which full-length Ath1 fused
to reporter mCherry was barely detected, whereas a partially proteolysed Ath1 fragment was predomi-nantly observed Also, use of a vps4D strain impaired Ath1 delivery to the vacuole and significantly reduced its proteolysis Similar observations were obtained with the vps1D strain (S He, unpublished), which was ini-tially identified as a protein involved in transport from the late-Golgi complex to the prevacuolar compart-ment [31] in the vacuole protein-sorting pathway To summarize, these results demonstrated that the vacuole
Trang 10is not the obligate functional destination for Ath1, and
that partial proteolysis of Ath1 could take place in this
subcellular compartment In contrast, targeting this
enzyme at the cell surface is indispensable for growth
of yeast cells on trehalose
Ath1 domains relevant for cell-surface targeting
and protein function
The finding that Ath1 could be targeted at the cell
periphery raised questions about secretion
determi-nants because domain-predicting tools did not identify
any sequence feature to explain Ath1 intracellular
traf-ficking Klionsky and co-workers [12] showed that the
short TM domain located at the N-terminus of Ath1
contained sufficient information to deliver Ath1 to the
vacuole via the MVB pathway They reached this
con-clusion using a chimeric construct in which only the
TM domain was fused to GFP Alternatively, we
spe-cifically removed the unique TM domain from
full-length Ath1 or from the 131 amino acid N-terminal
fragment fused to Suc2, and found that absence of this
TM domain abrogated the activity of invertase and
growth on sucrose More remarkably, removal of TM
in Ath1 led to a complete loss of enzyme activity and
the inability of a HA antibody to detect the HA–
Ath1DTM construct Because we were able to verify
that the absence of Ath1 protein was not caused by
inefficient ATH1 transcription (not shown), these
results suggested a critical function for the TM domain
in the translation and⁄ or stabilization of Ath1 during
early secretion steps This also fits with the
mislocaliza-tion of the NDTM–mCherry chimera in cytosolic
patchy bodies, whose origin is currently unknown
As indicated by hybrid Ath1–invertase fusions, a
131 amino acid N-terminal fragment was needed to
recover normal invertase secretion, whereas reducing
this N-terminal fragment to only 69 amino acids
decreased the secretion and activity of invertase at the
cell surface Taking this result together with those
using the reporter protein mCherry, the minimal infor-mation for correct targeting to the cell surface is likely localized between amino acids 69 (after the TM domain) and 131 of Ath1 protein sequence Several intracellular enzymes in yeast, in particular the glyco-lytic enzymes glyceraldehyde dehydrogenase [32], 3-phosphoglycerate mutase [33] and enolase [34,35], were found to be secreted at the cell surface although they did not harbor any classical signal sequence for secretion Nombela et al [36] proposed that these sig-nalless proteins could be exported by nonclassical export systems, such as those identified in mammals and parasites, which involve membrane blebbing (bub-ble formation) and secondary-structure elements that might also contribute to export [37] A common fea-ture between these glycolytic enzymes and S cerevisiae Ath1 is the lack of a classical secretion sequence How-ever, because Ath1 is not a cytosolic protein, these modes of secretion remain unknown By contrast, the classical secretion pathway cannot be excluded because
it was reported that mutations that cause accumulation
of secretory proteins in the endoplasmic reticulum (sec18) or in the Golgi apparatus (sec7) led to dimin-ished Ath1 activity [38,39] Also, previous findings of co-purification of Ath1 with cell-surface secreted pro-teins such as invertase [7,40] and Ygp1 [41] further supported this mode of secretion In conclusion, the secretion pathway for Ath1 needs to be thoroughly reinvestigated using specific mutants altered in various secretion processes
Experimental procedures
Strains, media and culture conditions
BY4741 (MAT a his3-D1 leu2-D0 ura3-D0 met15-D0), BY4742 (MAT a his3-D1 leu2-D0 lys2-D0 ura3-D0) and SEY6210 (MAT a his3-D200 leu2-3,112 lys2-801 trp1-901 ura3-52 suc2-D9) were used as recipient strains for various gene constructs, as described in Table 1 Yeast transforma-Table 1 Strains used in this study Euroscarf, Institute of Molecular Biosciences, Johann Wolfgang Goethe-University Frankfurt, Germany;
H Bussey, McGill University, Que´bec, Canada.
BY4741 vps4D ATH1_mCherry MAT a his3-D1 leu2-D0 ura3-D0 met15-D0 vps4D::KanMX4 ATH1-mCherry-His3MX6 This study