Activation of the plasma membrane H+-ATPase ofSaccharomyces cerevisiae by glucose is mediated bydissociation of the H+-ATPase–acetylated tubulin complexAlexis N. Campetelli1, Gabriela Previtali1, Carlos A. Arce2, Hector S. Barra2 and Cesar H. Casale1

1 Departamento de Biologıa Molecular, Facultad de Ciencias Exactas, Fısico-Quımicas y Naturales, Universidad Nacional de Rıo Cuarto,

Cordoba, Argentina

2 Centro de Investigaciones en Quımica Biologica de Cordoba (CIQUIBIC), UNC-CONICET, Departamento de Quımica Biologica,

Facultad de Ciencias Quımicas, Universidad Nacional de Cordoba, 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 Quımica

Biologica, Facultad de Ciencias Quımicas,

Universidad Nacional de Cordoba, Ciudad

Universitaria, 5000-Cordoba, 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.

5742 FEBS Journal 272 (2005) 5742–5752 ª 2005 FEBS

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 of D-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

A B

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 mM 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.

A. N. Campetelli et al. Dissociation of tubulin–H+-ATPase complex by glucose

FEBS Journal 272 (2005) 5742–5752 ª 2005 FEBS 5743

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 experiments.

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 No addition (control) 100 ± 11 1.49 ± 0.6

0.1 mM D-glucose 20 ± 7 8.42 ± 0.27

0.1 mM D-glucose plus 82 ± 9 2.21 ± 0.25

1 mM 2-deoxy-D-glucose

10 mM D-glucose plus 29 ± 13 5.72 ± 0.5

1 mM 2-deoxy-D-glucose

2 No addition (control) No addition 100 ± 9 1.39 ± 0.5

0.1 mM D-glucose plus

1 mM 2-deoxy-D-glucose

10 mM D-glucose 28 ± 12 5.60 ± 0.7

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 mM 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 mM 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.

Dissociation of tubulin–H+-ATPase complex by glucose A. N. Campetelli et al.

5744 FEBS Journal 272 (2005) 5742–5752 ª 2005 FEBS

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 (isolated 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).

A. N. Campetelli et al. Dissociation of tubulin–H+-ATPase complex by glucose

FEBS Journal 272 (2005) 5742–5752 ª 2005 FEBS 5745

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 mM 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 mM 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 mM Tris ⁄HCl buffer,pH 7.4, containing 150 mM 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.

Dissociation of tubulin–H+-ATPase complex by glucose A. N. Campetelli et al.

5746 FEBS Journal 272 (2005) 5742–5752 ª 2005 FEBS

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 proportions 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 measuring 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 mM 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.

A. N. Campetelli et al. Dissociation of tubulin–H+-ATPase complex by glucose

FEBS Journal 272 (2005) 5742–5752 ª 2005 FEBS 5747

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)1 protein. Acetylated tubulin bands shown

are from a representative experiment.

Dissociation of tubulin–H+-ATPase complex by glucose A. N. Campetelli et al.

5748 FEBS Journal 272 (2005) 5742–5752 ª 2005 FEBS

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-

logıa 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.Ænmol)1) and 7 lgÆmL)1 plasma membrane protein. After

20 min at 30 �C, the reaction was stopped by adding 50 lLof 66% (w ⁄ v) trichloroacetic acid per mL of incubation

mixture, and the released 32Pi was quantified. Plasma mem-

brane H+-ATPase activity was calculated as the difference

A. N. Campetelli et al. Dissociation of tubulin–H+-ATPase complex by glucose

FEBS Journal 272 (2005) 5742–5752 ª 2005 FEBS 5749

of ATP hydrolysis in the presence vs. absence of 100 lmsodium 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, thepreparation 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

anti-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 �Cin 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 proportions 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)

Dissociation of tubulin–H+-ATPase complex by glucose A. N. Campetelli et al.

5750 FEBS Journal 272 (2005) 5742–5752 ª 2005 FEBS

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 Promocion Cientıfi-

ca y Tecnologica de la Secretarıa de Ciencia y Tec-

nologıa del Ministerio de Cultura y Educacion en el

marco del Programa de Modernizacion Tecnologica

(BID 802 OC ⁄AR), Fundacion Antorchas, Secretarıa

de Ciencia y Tecnica de la Universidad Nacional de

Cordoba, Secretarıa de Ciencias de la Universidad

Nacional de Rıo Cuarto, y Agencia Cordoba Ciencia

del Gobierno de la Provincia de Cordoba,

Argentina.

References

1 Alonso AC, Nunez-Fernandez M, Beltramo DM,

Casale CH & Barra HS (1998) Na+,K+-ATPase was

found to be the membrane component responsible for

the hydrophobic behaviour of the brain membrane tub-

ulin. Biochem Biophys Res Commun 253, 824–927 [note:

erratum published in Biochem. Biophys Res Commun

257 (1999), 642].

2 Casale CH, Alonso A del C & Barra HS (2001) Brain

plasma membrane Na+,K+-ATPase is inhibited by

acetylated tubulin. Mol Cell Biochem 216, 85–92.

3 Casale CH, Previtali G & Barra HS (2003) Involvement of

acetylated tubulin in the regulation of Na+,K+-ATPase

activity in cultured astrocytes. FEBS Lett 534, 115–118.

4 Casale CH, Previtali G, Serafino JJ, Arce CA & Barra

HS (2005) Regulation of acetylated tubulin ⁄Na+,K+-

ATPase interaction by l-glutamate in non-neural cells:

involvement of microtubules. Biochim Biophys Acta

1721, 185–192.

5 Serrano R (1983) In vivo glucose activation of the yeast

plasma membrane ATPase. FEBS Lett 156, 11–14.

6 Malpartida F & Serrano R (1981) Phosphorylated inter-

mediate of the ATPase from the plasma membrane of

yeast. Eur J Biochem 116, 413–417.

7 Capieaux E, Vignais M-L, Sentenac A & Goffeau A

(1989) The yeast H+-ATPase gene is controlled by the

promoter binding factor TUF. J Biol Chem 264, 7437–

7446.

8 Eraso P & Portillo F (1994) Molecular mechanism of

regulation of yeast plasma membrane H(+)-ATPase

by glucose. Interaction between domains and identifi-

cation of new regulatory sites. J Biol Chem 269,

10393–10399.

9 Garcia-Arranz M, Maldonado AM, Mazon MJ &

Portillo F (1994) Transcriptional control of yeast

plasma membrane H(+)-ATPase by glucose. Cloning

and characterization of a new gene involved in this

regulation. J Biol Chem 269, 18076–18082.

10 Souza MAA, Tropia MJ & Brandao RL (2001) New

aspects of the glucose activation of the H+-ATPase in

the yeast Saccharomyces cerevisiae. Microbiology 147,

2849–2855.

11 de la Fuente N, Maldonado AM & Portillo F (1997)

Glucose activation of the plasma membrane H+-ATP

ase requires the ubiquitin-proteosome proteolytic path-

way. FEBS Lett 411, 308–312.

12 Coons DM, Boulton RB & Bisson LF (1995) Compu-

ter-assisted nonlinear regression analysis of the multi-

component glucose uptake kinetics. J Bacteriol 177,

3251–3258.

13 Nunez-Fernandez M, Beltramo DM, Alonso A del C

& Barra HS (1997) Conversion of hydrophilic tubulin

into a hydrophobic compound. Evidence for the invol-

vement of membrane proteins. Mol Cell Biochem 170,

91–98.

14 Beltramo DM, Nunez M, Alonso A del C & Barra HS

(1994) The relationship of hydrophobic tubulin with

membranes in neural tissue. Mol Cell Biochem 141,

57–63.

15 Goossens A, de La Fuente N, Forment J, Serrano R &

Portillo F (2000) Regulation of yeast H+-ATPase by

protein kinases belonging to a family dedicated to acti-

vation of plasma membrane transporters. Mol Cell Biol

20, 7654–7661.

16 Villalba JM, Palmgren MG, Berberian GE, Ferguson C

& Serrano R (1992) Functional expression of plant

plasma membrane H(+)-ATPase in yeast endoplasmic

reticulum. J Biol Chem 267, 12341–12349.

17 Laemmli UK (1970) Cleavage of structural proteins

during the assembly of the head of bacteriophage T4.

Nature 227, 680–685.

18 Piperno G & Fuller MT (1985) Monoclonal antibodies

specific for an acetylated form of alpha-tubulin recog-

nize the antigen in cilia and flagella from a variety of

organisms. J Cell Biol 101, 2085–2094.

A. N. Campetelli et al. Dissociation of tubulin–H+-ATPase complex by glucose

FEBS Journal 272 (2005) 5742–5752 ª 2005 FEBS 5751

19 DeWitt ND, dos Santos CF, Allen KE & Slayman CW

(1998) Phosphorylation region of the yeast plasma-

membrane H+-ATPase. Role in protein folding and

biogenesis. J Biol Chem 273, 21744–21751.

20 Hubert JJ, Schenk DB, Skelly H & Leffert HL (1986) Rat

hepatic (Na+,K+)-ATPase: alpha-subunit isolation by

immunoaffinity chromatography and structural analysis

by peptide mapping. Biochemistry 5, 4156–4163.

21 Bradford MM (1976) A rapid and sensitive method for

the quantitation of microgram quantities of protein util-

izing the principle of protein-dye binding. Anal Biochem

72, 248–254.

5752 FEBS Journal 272 (2005) 5742–5752 ª 2005 FEBS

Dissociation of tubulin–H+-ATPase complex by glucose A. N. Campetelli et al.