Báo cáo khoa học: Activation of the plasma membrane H+-ATPase of Saccharomyces cerevisiae by glucose is mediated by dissociation of the H+-ATPase–acetylated tubulin complex docx

When cells were treated with glucose, the H+ -ATP-ase–tubulin complex was disrupted, with two consequences, namely a the level of acetylated tubulin in the plasma membrane decreased as a

Saccharomyces cerevisiae by glucose is mediated by

Alexis N Campetelli1, Gabriela Previtali1, Carlos A Arce2, He´ctor S Barra2and Ce´sar H Casale1

1 Departamento de Biologı´a Molecular, Facultad de Ciencias Exactas, Fı´sico-Quı´micas y Naturales, Universidad Nacional de Rı´o Cuarto, Co´rdoba, Argentina

2 Centro de Investigaciones en Quı´mica Biolo´gica de Co´rdoba (CIQUIBIC), UNC-CONICET, Departamento de Quı´mica Biolo´gica,

Facultad de Ciencias Quı´micas, Universidad Nacional de Co´rdoba, Argentina

We recently described the interaction of Na+⁄ K+-ATP

ase with acetylated tubulin in neural [1–3] and

non-neural cells [4] Formation of such a complex inhibits

ATPase activity Conversely, dissociation of the com-plex leads to activation of the enzyme The ATPase– acetylated tubulin complex behaves as a hydrophobic

Keywords

glucose; H + -ATPase; proton pump; tubulin;

yeast

Correspondence

H S Barra, Departamento de Quı´mica

Biolo´gica, Facultad de Ciencias Quı´micas,

Universidad Nacional de Co´rdoba, Ciudad

Universitaria, 5000-Co´rdoba, Argentina

Fax: +54 3514334074

Tel: +54 3514334168

E-mail: hbarra@dqb.fcq.unc.edu.ar

(Received 29 July 2005, revised 2 September

2005, accepted 6 September 2005)

doi:10.1111/j.1742-4658.2005.04959.x

In the yeast Saccharomyces cerevisiae, plasma membrane H+-ATPase is activated by d-glucose We found that in the absence of glucose, this enzyme forms a complex with acetylated tubulin Acetylated tubulin usu-ally displays hydrophilic properties, but behaves as a hydrophobic com-pound when complexed with H+-ATPase, and therefore partitions into

a detergent phase When cells were treated with glucose, the H+ -ATP-ase–tubulin complex was disrupted, with two consequences, namely (a) the level of acetylated tubulin in the plasma membrane decreased as a function of glucose concentration and (b) the H+-ATPase activity increased as a function of glucose concentration, as measured by both ATP-hydrolyzing capacity and H+-pumping activity The addition of 2-deoxy-d-glucose inhibited the above glucose-induced phenomena, sug-gesting the involvement of glucose transporters Whereas total tubulin is distributed uniformly throughout the cell, acetylated tubulin is concentra-ted near the plasma membrane Results from immunoprecipitation experiments using anti-(acetylated tubulin) and anti-(H+-ATPase) immu-noglobulins indicated a physical interaction between H+-ATPase and acetylated tubulin in the membranes of glucose-starved cells When cells were pretreated with 1 mm glucose, this interaction was disrupted Dou-ble immunofluorescence, observed by confocal microscopy, indicated that

H+-ATPase and acetylated tubulin partially colocalize at the periphery

of glucose-starved cells, with predominance at the outer and inner sides

of the membrane, respectively Colocalization was not observed when cells were pretreated with 1 mm glucose, reinforcing the idea that glucose treatment produces dissociation of the H+-ATPase–tubulin complex Biochemical experiments using isolated membranes from yeast and purified tubulin from rat brain demonstrated inhibition of H+-ATPase activity by acetylated tubulin and concomitant increase of the H+-ATP ase–tubulin complex

Abbreviations

HAT, hydrophobic acetylated tubulin.

compound, whereas free tubulin is soluble in water.

This property allowed us to isolate the complex, termed

hydrophobic acetylated tubulin (HAT), which was

subsequently quantified by immunoblot with antibody

specific to acetylated tubulin

The present study is focused on the H+-ATPase of

yeast plasma membrane, another P-type ATPase This

enzyme is formed by several polypeptides, most

prom-inently a 100 kDa chain that is partly immersed in the

plasma membrane It functions to hydrolyze ATP and

to transport H+ out of the cell, thereby regulating

internal pH An important finding was that glucose

activates the plasma membrane H+-ATPase of yeast

cells [5] This activation has been extensively

investi-gated; however, its molecular mechanism is not

com-pletely understood

The activation of yeast H+-ATPase by glucose is

regulated at the transcriptional and

post-transcrip-tional levels [6–10] Glucose increases mRNA synthesis

by inducing transcription of the H+-ATPase gene

(PMA1p), increases phosphorylation of the enzyme,

the Km decreases and the Vmax increases Proteolytic

degradation of a protein that inhibits the

glucose-activation process also seems to be involved [11] We

report here that yeast H+-ATPase interacts with

tubu-lin, that such an interaction inhibits enzyme activity,

and that glucose treatment of cells induces dissociation

of the ATPase–tubulin complex with a concomitant

increase in the amount of active enzyme

Results

Effect ofD-glucose on H+-ATPase activity and HAT quantity

Yeast cells suspended in Mes-Tris buffer were incuba-ted for 20 min at 30
C in the presence of various concentrations of d-glucose Plasma membranes were isolated, and H+-ATPase activity and HAT quantity were measured d-glucose produced concentration-dependent activation of H+-ATPase, as measured by its ATP-hydrolyzing capacity, reaching a plateau at

150 lm (Fig 1A) At this concentration, HAT was decreased by 80% Activation of plasma H+-ATPase

by d-glucose is a very rapid process; when the yeast cells were incubated in unbuffered medium in the pres-ence of 1 mm d-glucose, the level of HAT decreased to almost zero in less than 5 min (Fig 1B) Acidification

of the medium was observed, with a minimal pH value reached in less than 10 min The ATP-hydrolysis capa-city of H+-ATPase also increased quickly It should be noted that dissociation of the tubulin–H+-ATPase complex (initial rate of decay of HAT) precedes enzyme activation determined by its H+-pumping or ATP hydrolyzing activity (Fig 1B) These rapid effects of

d-glucose suggest action at the membrane level, poss-ibly during the transport of glucose into the cell We therefore studied the effect of 2-deoxy-d-glucose, a competitive substrate for glucose uptake [12] This

Fig 1 Effect of D -glucose on H+-ATPase activity and hydrophobic acetylated tubulin (HAT) quantity in the plasma membrane of Saccharomy-ces cerevisiae (A) Glucose-starved yeast cells were incubated for 20 min at 30
C in Mes ⁄ Tris buffer in the presence of D -glucose at the indicated concentrations (B) Yeast cells were incubated at 30
C in physiological solution in the presence of 1 m M D -glucose for the indica-ted times Cells were processed for membrane isolation and subsequent determination of H+-ATPase activity and HAT quantity, as des-cribed in the Experimental procedures (B) In a parallel experiment, at the indicated time-points the external pH was measured instead of proceeding to membrane isolation Data represent the mean ± SD from three independent experiments Acetylated tubulin bands shown in the upper panels are from a representative experiment.

compound, at a concentration of 1 mm in the culture

medium, blocked the H+-ATPase activation and HAT

decrease induced by 30 min treatment of cells with

0.1 mm d-glucose (Table 1, Exp 1); however, it had no

effect when used at 10 mm The blocking of the

ATP-ase activating effect of glucose was not the result of a

toxic effect of 2-deoxy-d-glucose, as subsequent

incuba-tion in the presence of 10 mm glucose resulted again in

H+-ATPase activation with concomitant dissociation

of the ATPase–tubulin complex (HAT decrease)

(Table 1, Exp 2) The blocking effect of

2-deoxy-d-glucose is immediate as, 1 min after its addition, the

H+-pumping activity of H+-ATPase ceased (Fig 2A)

and the HAT quantity began to increase (Fig 2B)

When isolated plasma membranes from

glucose-starved yeast were used instead of intact cells,

treat-ment with 1 mm d-glucose for 20 min had no effect on

the ATP-hydrolyzing capacity of H+-ATPase, nor on HAT quantity (data not shown)

Previous studies [2–4], and the findings described below, suggest that the decrease of HAT in the plasma membrane should be interpreted as a lower level of the

H+-ATPase–tubulin complex We therefore presume that the immediate uptake of glucose after its addition induces dissociation of the H+-ATPase–acetylated tub-ulin complex, resulting in an increased enzyme activity, characterized by higher ATPase hydrolyzing capacity and H+-pumping activity

Characterization of acetylated tubulin in Saccharomyces cerevisiae

Localization of acetylated tubulin in cells was studied

by immunofluorescence Acetylated tubulin (Fig 3A)

Table 1 Effect of 2-deoxy- D -glucose on H + -ATPase activation induced by D -glucose Glucose-starved cells suspended in Mes-Tris buffer were incubated at 37
C under the indicated conditions for 30 min (experiment 1) or 60 min (experiment 2), then frozen in liquid nitrogen, and hydrophobic acetylated tubulin (HAT) and H+-ATPase activities were measured Values represent the mean ± SD from three independ-ent experimindepend-ents.

Experiment

Additions and

30 min of incubation

Additions and 30 min

of further incubation

HAT (% of control)

H + -ATPase activity (lmol PiÆmin)1Æmg protein)1)

1 m M 2-deoxy- D -glucose

1 m M 2-deoxy- D -glucose

0.1 m M D -glucose plus

1 m M 2-deoxy- D -glucose

A

B

Fig 2 Effect of 2-deoxy- D -glucose on H + -pumping activity and hydrophobic acetylated tubulin (HAT) quantity of Saccharomyces cerevisiae (A) Yeast cells were incubated at 30
C in physiological solution in the presence of 1 m M D -glucose, and the external pH was measured at the indicated time-points At the time indicated by the arrow, 2-deoxy- D -glucose (10 m M final concentration) was added (d) A control, with-out added 2-deoxy- D -glucose, was included (s) (B) In a parallel experiment, instead of pH measurement, HAT quantity was measured Data represent the mean ± SD from three independent experiments Acetylated tubulin bands shown are from a representative experiment.

and H+-ATPase (Fig 3C) are localized near the

plasma membrane, whereas a-tubulin (Fig 3B) is

dis-tributed uniformly throughout the cell

We showed previously [13,14] that the presence of acetylated tubulin in membrane preparations from mammalian cells is the result not of an intrinsic prop-erty of the protein, but because of its association with

an integral membrane protein subsequently identified

as Na+⁄ K+-ATPase [1] Acetylated tubulin present in the membrane was released by incubation at alkaline

pH (0.1 m Na2CO3, pH 11.5) and the remaining mem-branes (depleted of acetylated tubulin) could again accept exogenously added acetylated tubulin [14] We therefore investigated the biochemical properties of acetylated tubulin in yeast plasma membrane, and found them to be similar to those of acetylated tubulin

in mammalian cells After alkaline treatment, yeast plasma membrane was depleted of total tubulin (deter-mined with DM1A antibody) as well as acetylated tubulin (determined with 6-11B-1 antibody) (Fig 4, lane 2 vs lane 1) When acetylated tubulin-depleted membranes were incubated in the presence of

acetylat-ed tubulin (isolatacetylat-ed from rat brain), this protein was bound to the membrane fraction (lane 3) and subse-quently removed by alkaline treatment (lane 4) These results indicate that acetylated tubulin in yeast

Fig 3 Immunofluorescent localization of total microtubules,

acetyl-ated tubulin, and H + -ATPase in Saccharomyces cerevisiae

Sphero-plasts from glucose-starved yeast cells were fixed on coverslips

and stained with antibody against acetylated tubulin (A), total

a-tub-ulin (B), or H + -ATPase (C), using corresponding secondary

antibod-ies conjugated to fluorescein for acetylated and total a-tubulin and

rhodamine for H+-ATPase.

Fig 4 Removal of acetylated tubulin from yeast membranes by alkaline treatment Plasma membranes (5 mg of protein) from yeast (lane 1) were suspended in 5 mL of 0.1 M Na2CO3, pH 11.5, and incubated for 20 min at 4
C The sample was centrifuged at

100 000 g for 20 min, and the pellet (lane 2) was resuspended in Mes ⁄ Tris buffer, pH 6.5 This preparation (2 mg of protein) was incubated for 20 min at 37
C with partially purified brain tubulin (2 mg) containing a high proportion of acetylated isotype The pre-paration was centrifuged at 100 000 g for 20 min, and the pellet (lane 3) was collected The pellet was treated with alkaline solution

as described above, and centrifuged to sediment the membrane fraction (lane 4) The starting plasma membrane from yeast, and the subsequent pellets, were processed to determine hydrophobic acetylated tubulin (HAT) by immunoblot, as described in the Experi-mental procedures, using antibody to total a-tubulin (left panel) and

to acetylated tubulin (right panel).

membrane is a peripheral protein that is bound to

some integral membrane protein, presumably H+

-ATP-ase, in a manner similar to its association with

Na+⁄ K+-ATPase in the plasma membrane of

mamma-lian cells

To confirm the interaction of acetylated tubulin with

H+-ATPase and the presence of the H+

-ATPase–acet-ylated tubulin complex in membranes of glucose-starved

yeast, we performed immunoprecipitation experiments

using either anti-(acetylated tubulin) immunoglobulin

or anti-(H+-ATPase) immunoglobulin bound to

Seph-arose beads When detergent-solubilized plasma

mem-branes from glucose-starved yeast were treated with

anti-(acetylated tubulin)–Sepharose beads and then

cen-trifuged, in addition to tubulin, the pellet contained the

110 kDa H+-ATPase polypeptide (Fig 5B, lane –)

When solubilized membranes were treated with

anti-H+-ATPase–Sepharose beads, the pellet contained

acet-ylated tubulin in addition to the 110 kDa H+-ATPase

polypeptide (Fig 5C, lane –) These findings indicate

that a complex containing both acetylated tubulin and

H+-ATPase is present in solubilized yeast membranes

from glucose-starved cells When cells were pretreated

with 1 mm glucose for 1 h and then processed for

im-munoprecipitation with anti-H+-ATPase–Sepharose

beads, the pellet contained the 110 kDa H+-ATPase

polypeptide and only a minor amount of acetylated

tub-ulin (Fig 5C, lane +), indicating that glucose treatment

induced dissociation of the H+-ATPase–acetylated

tub-ulin complex An identical conclusion was drawn from

immunoprecipitation experiments with anti-(acetylated

tubulin)–Sepharose beads (Fig 5B, lane +) Figure 5D

shows that Sepharose beads without bound antibodies

practically do not adsorb tubulin or ATPase To

esti-mate the proportion of acetylated tubulin and H+

-ATP-ase that was immunoprecipitated, we determined also

the amount of each protein present in the

solubilized-membrane preparations before immunoprecipitation

(Fig 5A) From visual comparison of blots, it can be

seen that most of the acetylated tubulin and ATPase

were involved in the immunoprecipitation process By

measuring the intensity of the ATPase band in Fig 5B

(lane –) with respect to that in Fig 5A (lane –), it was

calculated that 88 ± 15% of ATPase was associated

with acetylated tubulin In addition, by comparing the

intensity of the ATPase band in lane (+) with respect to

that in lane (–) in Fig 5B, it was calculated that

88 ± 12% of the complex was dissociated by glucose

treatment These results represent the mean ± SD

val-ues from three independent experiments

Further evidence for the occurrence of the H+

-ATP-ase–acetylated tubulin complex in the plasma

mem-brane of S cerevisiae was the co-localization of the

two components in confocal immunofluorescence ana-lysis The image obtained with anti-(acetylated tubulin) immunoglobulin partially overlapped that obtained with anti-(H+-ATPase) immunoglobulin (Fig 6) Enlargement of a portion of the merge image (Fig 6D) shows that H+-ATPase (red) is localized on the plasma membrane without extending into the cyto-plasm, while acetylated tubulin (green) is localized near the membrane, overlapping (yellow) with the inner side

of the region occupied by H+-ATPase The more external region of the membrane H+-ATPase is not in contact with acetylated tubulin When cells were pre-treated with 1 mm glucose for 1 h and then processed for immunofluorescence, acetylated tubulin was distri-buted uniformly throughout the cytoplasm and did not

Fig 5 Physical interaction between acetylated tubulin and H + -ATP-ase in Saccharomyces cerevisiae membrane (A) To estimate the total amount of both acetylated tubulin and ATPase present in the detergent-solubilized yeast plasma membrane from cells previously treated (lanes +) or not treated (lanes –) with 1 m M glucose, 23 lL aliquots were subjected to SDS ⁄ PAGE and simultaneous immuno-blot staining with anti-(acetylated tubulin) and anti-(H + -ATPase) immunoglobulin (B) and (C) An aliquot of 0.7 mL of detergent-solubi-lized yeast plasma membrane from cells previously treated (lanes +)

or not treated (lanes –) with 1 m M glucose was mixed with 0.3 mL

of packed anti-acetylated tubulin (6-11-B-1)–Sepharose beads (B), or anti-H+-ATPase (Pma1p)–Sepharose beads (C), and incubated at

20
C for 30 min The samples were then centrifuged, and the preci-pitated material was washed five times with 50 m M Tris ⁄ HCl buffer,

pH 7.4, containing 150 m M NaCl (TBS)-Triton A fraction of 50 lL of packed beads was resuspended in 50 lL of Laemmli sample buffer [13], treated at 50
C for 15 min, and centrifuged Twenty microlitre aliquots of the soluble fractions were subjected to SDS ⁄ PAGE and then to simultaneous immunoblot staining with anti-(acetylated tubu-lin) and anti-(H + -ATPase) immunoglobulin (D) A control was run in parallel using glycine–Sepharose instead of antibodies–Sepharose Note that the protein bands shown arise from equal amounts of membrane preparations.

overlap ATPase, which remained concentrated at the

periphery This is further evidence that glucose induced

dissociation of the H+-ATPase–acetylated tubulin

complex

H+-ATPase activity of isolated plasma membrane

is inhibited by tubulin Plasma membranes isolated from S cerevisiae were incubated in the presence of various amounts of tubu-lin purified from rat brain, and H+-ATPase activity was determined Enzyme activity decreased as the tub-ulin concentration increased (Fig 7A) Two prepara-tions were used containing proporprepara-tions of acetylated tubulin isotype that differed approximately fourfold The amount of tubulin required to obtain 50% inhibi-tion of H+-ATPase was approximately fourfold higher for the low-proportion preparation For each tubulin concentration, we determined the amount of acetylated tubulin that was converted into hydrophobic com-pound (HAT) (Fig 7B) Such conversion reflects for-mation of the tubulin–ATPase complex The HAT quantity increased as the tubulin concentration increased Taken together, these results indicate that the association of H+-ATPase with acetylated tubulin inhibits enzyme activity

Discussion

In yeast, the H+-ATPase activity of the plasma mem-brane is up-regulated by external glucose [5–10] We showed, in this study, that tubulin interacts with

H+-ATPase to form a complex in which the enzyme is inhibited, and that treatment of the cells with glucose dissociates the complex and restores enzyme activity Although tubulin is a hydrophilic protein, it behaves

as a hydrophobic compound when it interacts with

H+-ATPase The complex can therefore be isolated by detergent-partitioning with Triton X-114 The hydro-phobic tubulin partitioning into the detergent phase (HAT) is the acetylated tubulin forming a complex with H+-ATPase

The finding of the H+-ATPase–acetylated tubulin complex in plasma membranes of glucose-starved yeast cannot be attributed to an in vitro artifact (i.e associ-ation of acetylated tubulin with H+-ATPase during the isolation of HAT) because a high vs a low content

of HAT is measured in glucose-untreated or -treated cells, respectively, by the same isolation procedure If the interaction between tubulin and H+-ATPase were established during the in vitro procedure, the amount

of HAT in membranes from glucose-treated cells would be the same as that from glucose-untreated cells The possibility that the presence of glucose dur-ing the in vitro procedure for measurdur-ing HAT induced dissociation of the ATPase–acetylated tubulin complex was also experimentally ruled out The treatment of isolated membranes with 1 mm glucose did not

Fig 6 Colocalization of acetylated tubulin and H + -ATPase in

Sac-charomyces cerevisiae Yeast cells were treated (+ glucose) or

were not treated (– glucose) with 1 m M glucose for 1 h and

proc-essed to obtain spheroplasts, which were then fixed on coverslips

and subjected to double immunofluorescence using antibodies

spe-cific to H + -ATPase (A and A¢) and to acetylated tubulin (B and B¢).

(C) and (C¢) Merge image (D) and (D¢) Enlargement of the area

indi-cated by a rectangle in (C) and (C¢), respectively.

decrease HAT or increase H+-ATPase activity (data

not shown)

The presence of the H+-ATPase–acetylated tubulin

complex in the plasma membrane of glucose-starved

yeast is also supported by results from

immunoprecipi-tation experiments (Fig 5) As acetylated tubulin

pre-sent in detergent-solubilized membranes isolated from

glucose-starved yeast was precipitated by treatment with

anti-(H+-ATPase) immunoglobulin (Fig 5C, lane –),

and H+-ATPase was precipitated by treatment with

anti-(acetylated tubulin) immunoglobulin (Fig 5B,

lane –), we conclude that a complex containing H+

-ATPase and acetylated tubulin is present in the plasma

membrane Colocalization of acetylated tubulin with

H+-ATPase in the periphery of the cell was observed by

confocal microscopy (Fig 6) Although the overlap was

partial, it is clear that some part of the acetylated

tubu-lin shares space with some part of the H+-ATPase,

rein-forcing the idea of a complex between H+-ATPase and

acetylated tubulin at the plasma membrane This

com-plex was dissociated by pretreatment of cells with

glu-cose In fact, when cells were treated with 1 mm glucose

for 1 h prior to immunoprecipitation and

immunofluo-rescence experiments, acetylated tubulin was diminished

in isolated membranes (Fig 5A, lane +), and

practi-cally was not precipitated by anti-(H+-ATPase)

immu-noglobulin (Fig 5C, lane +), and ATPase and

acetylated tubulin did not colocalize (Fig 6D¢)

We observed that treatment of cells with glucose

induces dissociation of the complex [seen as a decrease

of HAT, a decrease of acetylated tubulin

immuno-precipitated by anti-(H+-ATPase) immunoglobulin, a decrease of H+-ATPase immunoprecipitated by anti-tubulin immunoglobulin, and a loss of colocalization] with concomitant increase of H+-ATPase activity This provides strong evidence for existence of the

H+-ATPase–acetylated tubulin complex in the plasma membrane of the yeast cell, and for a regulatory role

of tubulin on activity of the enzyme

We often refer to ‘H+-ATPase–tubulin’ rather than

‘H+-ATPase–acetylated tubulin’ complex, for conveni-ence In fact, it is quite possible that acetylated tubulin

is the only tubulin isotype that forms the complex, as

H+-ATPase activity is more strongly inhibited when the tubulin preparation contains a higher proportion

of the acetylated isotype (Fig 7)

We do not know whether molecules other than

H+-ATPase and tubulin are also part of the complex Although the inhibition of H+-ATPase activity observed when the complex is formed suggests direct interaction of tubulin with ATPase, we cannot rule out the possibility that other molecules mediate this interac-tion Involvement of glucose transporters is a reason-able assumption because the complex is dissociated when glucose is transported into the cell, but not when glucose is added to previously isolated membranes (data not shown) This possibility is supported by the obser-vation that 2-deoxy-d-glucose, a competitive substrate for glucose uptake, suppressed activation of glucose and dissociation of the ATPase–tubulin complex in a con-centration-dependent manner (Table 1) It is possible that the dissociation of the acetylated tubulin–H+

-A

B

Fig 7 Effect of exogenous tubulin on H + -ATPase activity and hydrophobic acetylated tubulin (HAT) quantity in isolated membranes Plasma membrane (70 lg of proteinÆmL)1) isolated from glucose-treated Saccharomyces cerevisiae was incubated at 37
C for 25 min in a final vol-ume of 1 mL of Mes-Tris ⁄ phenylmethanesulfonyl fluoride buffer, pH 6.5, in the presence of various amounts of rat brain tubulin preparations containing low (d) or high (s) levels of the acetylated isotype (for details see the Experimental procedures) After incubation, appropriate aliquots were removed to determine H+-ATPase activity (A) and HAT quantity (B) Values represent the mean ± SD from three independent experiments H + -ATPase activity in the absence of added tubulin was 8.2 ± 0.3 lmol PiÆmin)1Æmg)1protein Acetylated tubulin bands shown are from a representative experiment.

ATPase complex requires the presence of Snf3p (a

glu-cose sensor), Gpa2 protein (a G protein) [10] and

protein kinases [15], which were demonstrated to

parti-cipate in the glucose-induced activation of the plasma

membrane ATPase

Our results in the present work show that a

glucose-sensitive association⁄ dissociation of acetylated tubulin

and H+-ATPase participates in an early stage of the

mechanism that leads to, respectively, inhibition and

activation of the enzyme The increase of enzyme

activity (determined by ATP-hydrolyzing capacity or

by H+-pumping activity) starts immediately after

glu-cose addition and reaches a maximum in  10 min,

whereas dissociation of the ATPase–tubulin complex

(decrease of HAT quantity) is completed within the

first 2 min (Fig 1B, and Fig 2A,B) The reason for

this temporal difference is not clear It is possible that

besides dissociation of the complex, the enzyme

requires some additional, time-consuming process for

its activation Anyway, the important conclusion from

these experiments is that dissociation of the ATPase–

tubulin complex is at least part of the reason for the

increased enzyme activity induced by external glucose

In agreement with this view, exogenously added

tubu-lin was bound to membrane H+-ATPase and inhibited

enzyme activity (Fig 7), indicating that H+-ATPase

activation by glucose is caused by an increased

concen-tration of active enzyme Similar effects of tubulin

were demonstrated for Na+⁄ K+-ATPase in our

previ-ous studies [2–4]

Interestingly, for both H+-ATPase in yeast and

Na+⁄ K+-ATPase in mammalian cells [3,4], the

ATPase–acetylated tubulin complex can be dissociated

during the uptake of d-glucose and l-glutamate,

respectively In either case, corresponding enzyme

activity increases upon dissociation of the complex

We suspect that these events are part of a signal

trans-duction cascade and are accordingly investigating the

nature of membranous and cytoplasmic components of

the system, interactions between components and

modulation of these interactions

Experimental procedures

Materials

Triton X-114, ATP, d-glucose, 2-deoxy-d-glucose, mouse

monoclonal antibody DM1A specific to a-tubulin, mouse

monoclonal antibody 6-11B-1 specific to acetylated tubulin,

anti-mouse and anti-rabbit IgG conjugated to peroxidase

were from Sigma Chemical Co (St Louis, MO, USA)

[32P]ATP[cP] was from Perkin-Elmer (Wellesley, MA,

USA) Rabbit polyclonal antibody Pma1p, specific to

H+-ATPase, was provided by R Serrano (Instituto de Bio-logı´a Molecular y Celular de Plantas, Valencia, Spain)

Yeast strain and growth conditions

S cerevisiae, strain CECT 1891 (Spanish Type Culture Collection, University of Valencia, Valencia, Spain), was used Cells were grown on synthetic medium YP [0.5% (w⁄ v) yeast extract and 0.5% (w ⁄ v) peptone] containing 4% (w⁄ v) glucose Cells were grown in a rotary incubator (New Brunswick model G24; 200 r.p.m.; NJ, USA) at

30
C until the end of the exponential phase Cells were then harvested (centrifugation at 1000 g for 10 min), sus-pended in Mes⁄ Tris buffer (100 mm Mes ⁄ Tris, pH 6.5) and magnetically stirred for 60 min to eliminate glucose activation [5] These cells are referred to as ‘glucose-starved cells’

Yeast plasma membrane preparation

Plasma membranes were isolated by a modification of the method of Villalba et al [16] Briefly, yeast cells were suspen-ded in Mes⁄ Tris buffer supplemented with 1 mm phenyl-methanesulfonyl fluoride (Mes⁄ Tris ⁄ phenylmethanesulfonyl fluoride buffer), with or without d-glucose, according to the conditions of each experiment, homogenized by vigorous shaking with glass beads, and centrifuged at 1000 g for

10 min The resulting supernatant was centrifuged at

70 000 g for 60 min to obtain the total membrane fraction The total membrane fraction from 10 g of cells was suspen-ded in 3 mL of Mes⁄ Tris ⁄ phenylmethanesulfonyl fluoride buffer The suspension was applied to a discontinuous gradi-ent – from 5.0 mL of 60% (w⁄ v) sucrose to 5.0 mL of 40% (w⁄ v) sucrose – in 10 mm Tris ⁄ HCl buffer (pH 7.6) contain-ing 1 mm EDTA and 1 mm dithiothreitol Plasma mem-branes were centrifuged for 3 h at 100 000 g, collected from the 40⁄ 60% sucrose interface, diluted 10-fold with Mes⁄ Tris ⁄ phenylmethanesulfonyl fluoride buffer and centri-fuged at 100 000 g for 1 h The resulting pellet was resus-pended in Mes⁄ Tris ⁄ phenylmethanesulfonyl fluoride buffer and stored at )70
C until use (maximum storage time

3 months)

Plasma membrane H+-ATPase activity assay

We used the [32P]ATP[cP] hydrolysis method of Malpartida

& Serrano [6] The incubation mixture (0.5 mL final vol-ume) contained Mes⁄ Tris ⁄ phenylmethanesulfonyl fluoride buffer, 10 mm MgCl2, 2 mm [32P]ATP[cP] (450 d.p.m.Æ nmol)1) and 7 lgÆmL)1 plasma membrane protein After

20 min at 30
C, the reaction was stopped by adding 50 lL

of 66% (w⁄ v) trichloroacetic acid per mL of incubation mixture, and the released32Piwas quantified Plasma mem-brane H+-ATPase activity was calculated as the difference

of ATP hydrolysis in the presence vs absence of 100 lm

sodium orthovanadate

Isolation and determination of HAT

HAT was isolated into the Triton X-114 phase, as

des-cribed previously [2], except that plasma membranes were

solubilized with Triton X-100, as described below, before

adding Triton X-114 and partitioning Membranes from

S cerevisiae (28 lg of protein) were washed once with

50 mm Tris⁄ HCl buffer, pH 7.4, containing 150 mm NaCl

(TBS) and immediately solubilized in 1 mL of TBS

con-taining 1% (v⁄ v) Triton X-100 After 30 min at 0
C, the

preparation was centrifuged at 100 000 g for 15 min, and

the supernatant fraction was processed for HAT isolation

by partitioning in Triton X-114 (1% final detergent

con-centration) For phase separation, the preparation was

warmed at 37
C for 5 min and centrifuged at 600 g for

5 min The aqueous upper phase and detergent-rich

lower phase were carefully separated, and the

detergent-rich phase (which contains HAT) was washed once

with TBS Aliquots were subjected to electrophoresis

and immunoblotting to determine acetylated and total

tubulin

Electrophoresis and immunoblotting

Proteins were separated by SDS⁄ PAGE on 10% (w ⁄ v)

polyacrylamide slab gels [17], transferred to nitrocellulose,

and reacted with mouse monoclonal antibody 6-11B-1

(dilution 1 : 1000) to determine acetylated tubulin [18],

mouse monoclonal antibody DM1A (dilution 1 : 1000) to

determine total a-tubulin, or rabbit polyclonal antibody

Pma1p (dilution 1 : 1000) to determine plasma membrane

H+-ATPase [15] The nitrocellulose sheet was reacted with

mouse (for 6-11B-1 and DM1A antibodies) or

anti-rabbit (for Pma1p antibody) IgG conjugated with

peroxi-dase Intensities of tubulin bands were quantified by Scion

imaging software

Measurement of H+-pumping activity

Samples of cells ( 50 mg) were washed twice, suspended

in 15 mL of 0.9% (w⁄ v) NaCl, and stirred gently at 30
C

in a beaker Incubation conditions were as indicated in each

experiment External pH was measured using a pH meter

with a calomel electrode

Preparation of spheroplasts from yeast

S cerevisiae cells (50 mg fresh weight) were washed twice

in 2 mL of 0.1 m Tris⁄ HCl, pH 7.2, containing 5 mm

EGTA and 5 mm dithiothreitol, incubated with stirring

for 10 min, washed with water (containing 1 mm

dithio-threitol), and resuspended in 2 mL of 0.1 m Tris⁄ HCl,

pH 7.2, containing 1 m sorbitol and 1 mm dithiothreitol Zymolyase was added to the suspension (final concentra-tion 0.1 mgÆmL)1), gently stirred for 30 min, and cells were harvested by centrifugation The pellet was resus-pended in 2 mL of 0.1 m Tris⁄ HCl, pH 7.2, containing

5 mm EGTA and 5 mm dithiothreitol, and used immedi-ately All procedures were carried out at room tempera-ture

Immunofluorescence

Spheroplasts were fixed with anhydrous methanol at )20
C on coverslips Samples were washed, incubated with 2% (w⁄ v) BSA in NaCl ⁄ Pi (PBS) for 30 min, and stained by indirect immunofluorescence, as described by DeWitt et al [19] Two primary antibodies were used: mouse 6-11B-1 monoclonal antibody (diluted 1 : 200) to visualize acetylated tubulin, and rabbit Pma1p polyclonal antibody (diluted 1 : 200) to determine plasma membrane

H+-ATPase Fluorescein-conjugated anti-mouse IgG and Rhodamine-conjugated anti-rabbit immunoglobulin, at a

1 : 400 dilution, were used as secondary antibodies, respectively Coverslips were mounted in FluorSave and observed with an LSM 5 Pascal confocal microscope (Zeiss, Jena, Germany) using dual channel filters for sim-ultaneous viewing of rhodamine and fluorescein isothio-cyanate fluorochromes

Tubulin preparations containing different proportions of acetylated tubulin

The procedure used to isolate rat brain tubulin prepara-tions containing different proporprepara-tions of the acetylated iso-type has been described previously [2] These preparations contained low and high acetylated tubulin proportions dif-fering by a factor of 4

Preparation of 6-11B-1–Sepharose and Pma1p– Sepharose

6-11B-1 and Pma1p antibodies were covalently bound to cyanogen bromide-activated Sepharose 4B following the procedure of Hubert et al [20] with slight modifications Sepharose beads were washed with a 100-fold volume excess of 0.001 m HCl at 21
C The resulting packed beads (0.3 mL) were mixed with 2 mg of 6-11B-1 antibody (or

2 mg of Pma1p antibody) in 1 mL of coupling buffer (0.5 m NaCl containing 0.2 m NaHCO3, pH 8.2) The mix-ture was agitated on a platform rocker for 4 h at 21
C, and loaded into a small chromatographic column Unbound 6-11B-1 antibody (or Pma1p antibody) was removed by filtration and by washing with 10 mL of coup-ling buffer The 6-11B-1-Sepharose (or Pma1p-Sepharose)

beads were loaded into a beaker, and added with 2 mL of

coupling buffer containing 0.2 m glycine to block unreacted

Sepharose sites The mixture was agitated for 2 h at 21
C,

and unbound glycine was removed by washing the beads

with 20 mL of coupling buffer The resulting affinity

adsorbant was washed with 30 mL of 0.01 m Tris⁄ HCl,

pH 8, containing 0.14 m NaCl and 0.025% NaN3, and

stored at 4
C until use (maximum 2 days)

Protein determination

Protein concentration was determined by the Bradford

method [21]

Acknowledgements

We thank Dr J A Curtino for critical reading of

the manuscript, Dr R Serrano (Valencia, Spain) for

the generous gift of anti-(H+-ATPase), and Dr S

An-derson for editing This work was supported by

grants from Agencia Nacional de Promocio´n

Cientı´fi-ca y Tecnolo´giCientı´fi-ca de la Secretarı´a de Ciencia y

Tec-nologı´a del Ministerio de Cultura y Educacio´n en el

marco del Programa de Modernizacio´n Tecnolo´gica

(BID 802 OC⁄ AR), Fundacio´n Antorchas, Secretarı´a

de Ciencia y Te´cnica de la Universidad Nacional de

Co´rdoba, Secretarı´a de Ciencias de la Universidad

Nacional de Rı´o Cuarto, y Agencia Co´rdoba Ciencia

del Gobierno de la Provincia de Co´rdoba,

Argentina

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