Determination of Pma1 activity in situ
The permeabilized cells were thawed at 28°C, washed 3 times with 5 ml of PB, resuspended in 2 ml of the same buffer, and stored at 0°C until use. A total of 20 µl of 100 mM Mg-ATP (Boehringer, USA), 1 µl of 10 µM Bafilomycin (Sigma, USA) in DMSO (for inhibition of the vacuolar ATPase), and 50 µl of cell suspension were added to 910 µl of PB. Then, 20 µl of 5 mM VO4
was added to the controls for the inhibition of Pma1. The reaction mixture was incubated at 30°C at 1,000 rpm in a Thermomixer compact (Eppendorf, FRG) for 30 min. The cells were precipitated at 14,000 g for 3 min, and the aliquots were diluted 50–100 times with ddH2
O. In a 96-well microplate, 200 µl of malachite green (Applechem, FRG) was added to 100 µl of a sample 
and incubated at 30°C for 15 min; the content of released Pi
was measured at 650 nm. Pma1 activity was calculated by the difference in the amount of inorganic Pi
released in the presence and absence of 100 µM sodium orthovanadate, the specific inhibitor of Pma1 
. To calculate the specific enzyme activity, total cell protein was determined 
with modifications. A total of 100 µl of the cell suspension in the PB was added to 1 ml of water and precipitated at 14,000 g. The precipitate was resuspended in 0.6 ml of water and 0.3 ml of 3 N NaOH was added and heated at 100°C for 5 min, followed by addition of 0.3 ml of 2.5% CuSO4
after cooling. Five minutes later, the mixture was centrifuged at 14,000 g, and the supernatant was measured by spectrophotometry at 555 nm. Specific Pma1 activity was expressed as nmol of inorganic Pi
released from ATP per minute per mg of total cell protein.
Results and Discussion
The association of Pma1 with lipid rafts has been studied quite thoroughly 
. However, little is known about the role of lipids in glucose activation of Pma1.
To shed light on this issue, we selected the mutant erg6
, which is deficient in ergosterol, and the mutant lcb1-100
, which is defective in sphingoid base synthesis. The incubation of prestarved cells with 100 mM glucose or 100 mM deoxyglucose for 15 min yielded the results shown in . As expected, both of the parent strains, BY4742
, demonstrated a glucose effect with different degrees of intensity. Unsurprisingly, the erg6
strain also showed glucose activation comparable with the parent strain. Previously, it was reported that disturbances in ergosterol synthesis in no way affected Pma1 biogenesis, secretion on the PM, or stability 
. In addition, ergosterol was shown to occur mainly in the membrane patches containing arginine and uracyl symporters but not Pma1 
In contrast, in strain lcb1-100
, incubation with glucose did not increase the enzyme activity and even slightly reduced it. At the same time, the basal Pma1 activity in strain lcb1-100
was somewhat higher than in the parent strain RH2874
. The latter result was quite unexpected, because previously it had been shown for the lcb1-100
strain that only a minor part of the newly synthesized Pma1 reaches PM at 30°C, whereas more than 90% of the enzyme is rerouted to the vacuole to be degraded 
. However, later work suggested that this effect depended in many respects on the cultivation medium 
. It should be noted that the growth of the lcb1-100
strain on the YPD medium was almost twice as low as that of the parent strain RH2874
(data not shown). With the exception of the erg6 s
train, deoxyglucose did not result in an increase in the Pma1 activity, in agreement with the Serrano's data 
Thus, we have shown that sphingolipid but not ergosterol is important for glucose activation of Pma1. This fact can be explained as follows: One of the consequences of sphingolipid synthesis disturbance in the lcb1-100
strain is inefficient or completely blocked Pma1 oligomerization 
, which probably results in the elimination of glucose activation. The difference in glucose effects on Pma1 activity in the erg6
strains may, therefore, be attributed to the sphingolipid associating with the protein at the very initial stages of biosynthesis of the enzyme and determining its oligomeric structure 
. Ergosterol, the other component of the lipid raft, appears not to participate directly in the formation of the oligomeric Pma1 complex and have no particular effect on the functioning of the protein. The idea that oligomerization of Pma1 is necessary for the glucose activation of Pma1 was indirectly confirmed in the earlier work 
. Using electron crystallography, researchers showed that the cytoplasmic part of Pma1 in a ligand-free form consists of four domains 
. Domain two of one Pma1 molecule directly contacts domain three of the neighboring molecule. Unfortunately, the authors of this work did not link these structural domains with the functional (ATP-binding, phosphorylation, C-terminal) domains. However, it may be hypothesized that in the absence of glucose, the nucleotide-binding domain of the Pma1 molecule is locked by the C-domain of the neighboring Pma1 molecule. In this case, glucose activation of the enzyme results in successive phosphorylation of Ser-911 and Thr-912, followed by the release of the C-tail from the nucleotide-binding domain, as demonstrated previously 
. Taking into account the intermolecular character of the described event, it may be supposed that Pma1 oligomerization is necessary for the activation of Pma1 by glucose.
Since the modern concept of glucose activation of Pma1 presupposes the movement of its C-tail 
, this process could be traced using the strain PMA1-GFP
, the Pma1 molecule of which carries a GFP domain at the C-terminus 
. To elucidate the effect of this spectral marker on glucose activation of the enzyme, the influence of the 15-min incubation of whole cells with 100 mM glucose or 100 mM deoxyglucose on Pma1 activity was investigated (). Both the parent strain SEY6210
and the strain PMA1-GFP
demonstrated a marked glucose effect that exceeded that of the BY4742
strains. Although the presence of the GFP domain resulted in a considerable decrease in basal Pma1 activity (18.5 and 7.3 nmol Pi
/min/mg total cell protein for SEY6210
, respectively), it had little effect on the Km
of the glucose-activated enzyme. The Km
values of Pma1 determined in the membrane fraction from glucose-activated cells of the SEY6210
strains were 0.22 mM and 0.41 mM, respectively. It is known that the Km
for glucose-activated Pma1 is 0.3–0.8 mM, in contrast to 2–4 mM for Pma1 in glucose-deprived cells 
. Thus, the PMA1-GFP
strain affords an opportunity to register the glucose activation process using modern spectral methods.
Fluorescence anisotropy was used to observe the structural rearrangements in the Pma1 molecules. The value of fluorescence anisotropy r
for the GFP monomer in the absence of substantial rotation of the fluorophore molecule during the fluorescence lifetime is close to the maximum value (0.4) 
. The radiationless transfer of energy between the fluorophore molecules (homo-FRET) as a result of oligomerization of the GFP molecules has been shown to result in a decrease in the observed effective value r
. shows the r
values of PMA1-GFP
whole cells in the absence and presence of (deoxy)glucose. Since the procedure for measuring the value of r
involves subtraction of the contribution of cellular autofluorescence to the total fluorescence, the observed r
value corresponds to that of the GFP molecules. As can be seen, the experimental r
values (0.165–0.202) were much lower than 0.38, the value r
for the monomeric and dimeric form of GFP expressed in different samples 
. The decrease in r
was typical of the homo-FRET phenomenon observed for the clusters of fluorophores approaching each other until the distance between them was less than 10 nm.
Fluorescence depolarization (anisotropy) r of PMA1-GFP in whole cells after 15 min incubation with 100 mM glucose or deoxyglucose.
Since it has been shown previously that the supramolecular Pma1complex in the PM consists of six units 
, the value r
in the absence of (deoxy)glucose (equal to 0.192) characterizes the anisotropy of the PMA1-GFP hexameric complex. The incubation of the PMA1-GFP
cells with 100 mM glucose for 15 min resulted in a marked decrease in the r
value by 0.026±0.005. The same incubation with deoxyglucose produced an insignificant opposite effect: an increase in the r
value by 0.011±0.006. Thus, there is a correlation between the values of Pma1 activity () and fluorescence anisotropy (). This finding implies that glucose activation of the enzyme is accompanied by certain structural rearrangements of the Pma1 molecules at the level of clusters. The following variants and their combinations are possible 
: a change in the number of PMA1-GFP molecules in a cluster or association of clusters with each other, a change in the average distance between the Pma1 molecules in a cluster, or regrouping/reorientation of the GFP domains without a change in the distance between the Pma1 molecules. The most probable explanation for the decrease in the r
value in our study seems to that since the activated enzyme intensively hydrolyzed ATP, the enzyme remained in the ligand-bound conformation for some of the time. Previously, it has been shown that the cytoplasmic Pma1 domain is more closely packed in this conformation 
. It has also been proposed that phosphorylation of Ser-911 and Thr-912 during glucose activation results in conformational changes in the Pma1 molecule in which the C-domain is released from the nucleotide-binding domain 
. In our model system, the C-tail of Pma1 carried a GFP domain. The decrease in the r
value may point to approaching of GFP domains within a hexamer complex during glucose activation of Pma1. However, in the case of a high fluorophore concentration, which often occurs in the case of membrane-bound proteins, the phenomenon of homo-FRET theoretically may be not a consequence of fluorophore oligomerization. Instead, it may be the result of the close approach of monomeric proteins (the so-called concentration depolarization effect) 
The change in the spatial distribution of the clusters containing Pma1 molecules during glucose metabolism was visualized using immunogold labeling. As shows, the distribution of the clusters of Pma1 molecules in the PM underwent substantial changes. In the glucose-starved cells of the parent strain SEY6210
, the distribution of the clusters of Pma1 molecules was relatively uniform, and the average distance between them exceeded 10 nm (). In the cells that had metabolized glucose, the clusters of Pma1 molecules aggregated in large groups of closely adjacent clusters (). Since immunogold labeling was performed on a cell cross-section but not on the inner leaflet of the PM obtained by freeze-fractioning, the size of the groups cannot be analyzed using Ripley's K-function. It is interesting that the approximate sizes of these groups of clusters of Pma1 molecules (about 70 nm) () are similar to the sizes of the individual lipid rafts (25–70 nm) 
. The distance between the Pma1 clusters in such a group was comparable to the distances at which the homo-FRET effect was exhibited (10 nm); therefore, the observed decrease in fluorescence anisotropy in response to glucose addition can be at least partially explained by this phenomenon. While intracellular labeling can be observed in all of the images, we assume that it is the labeling of the newly synthesized Pma1 molecules during their delivery to PM.
Immunogold labeling of Pma1 in the plasma membrane of S. cerevisiae SEY6210.
shows the effect of incubation with glucose on the spatial distribution of the Pma1 molecules in the PM of the erg6 and lcb1-100 strains. In the erg6 strain (), where glucose activated Pma1, the distribution of the clusters of enzyme molecules before and after the incubation with glucose was similar to the distribution of Pma1 in the SEY6210 strain. In the absence of glucose, Pma1 in the PM was distributed uniformly as separate particles. Following the incubation with glucose, Pma1 formed groups. In contrast to the SEY6210 and erg6 strains, the incubation with glucose did not change the distribution of Pma1 in the PM of the lcb1-100 strain (). The distribution of the Pma1 clusters remained uniform, without the formation of groups. Moreover, glucose did not exert an effect on the lcb1-100 strain (). Thus, it may be supposed that Pma1 activation by glucose is accompanied by the formation of groups of Pma1 clusters.
Immunogold labeling of Pma1 in the plasma membrane of S. cerevisiae erg6 and lcb1-100.
The revealed glucose-dependent movement of the clusters of the Pma1 molecules is noteworthy because it is a rather quick process (about 15 min). It is unclear what cellular mechanism underlies this reorganization of the Pma1 clusters, but one of the most probable reasons is the previously reported association of Pma1 with acetylated tubulin 
. In this study, the authors showed that Pma1 in the cells that did not metabolize glucose was associated with acetylated tubulin. On glucose addition, this complex very quickly degraded. These findings, together with our data presented above, suggest that acetylated tubulin in the absence of glucose fixes Pma1 clusters on the membrane and leads to a uniform distribution. Upon glucose addition, this complex disintegrates, allowing the clusters of Pma1 molecules to move freely in the membrane plane and combine into large groups.
These findings lead to the following two conclusions: (1) for glucose activation of Pma1 to take place, the enzyme is supposed to be in the oligomeric state, and (2) glucose activation is accompanied by the spatial movement of Pma1 clusters in the PM.