(Received for publication, September 27, 1994; and in revised form, January 4, 1995)
From the
The abundance of yeast plasma membrane H-ATPase
on the cell surface is tightly regulated. Modifier of pma1 (mop) mutants were isolated as enhancers of the mutant
phenotypes of pma1 mutants. mop2 mutations reduce the
abundance and activity of Pma1 protein on the plasma membrane without
affecting the abundance of other prominent plasma membrane proteins.
The MOP2 gene encodes a 108-kDa protein that has previously
been identified both as a gene affecting the yeast cytoskeleton (SLA2) (Holtzman, D.A., Yang, S., and Drubin, D. G.(1993) J. Cell Biol. 122, 635-644) and as a gene affecting
endocytosis (END4) (Raths, S., Roher, J., Crausaz, F., and
Riezman, H.(1993) J. Cell Biol. 120, 55-65). In some
strains, MOP2 (SLA2) is essential for cell viability;
in others, a deletion mutant is temperature sensitive for growth. mop2 mutations do not reduce the transcription of PMA1 nor do they lead to the accumulation of Pma1 protein in any
intracellular compartment. An epitope-tagged MOP2 protein
behaves as a plasma membrane-associated protein whose abundance is
proportional to its level of gene expression. Over-expression of MOP2 relieved the toxicity caused by the over-expression of PMA1 from a high copy plasmid; conversely, the growth of mop2 strains was inhibited by the presence of a single extra
copy of PMA1. We conclude that MOP2 (SLA2)
encodes a plasma membrane-associated protein that is required for the
accumulation and/or maintenance of plasma membrane
H
-ATPase on the cell surface.
The plasma membrane proton-translocating ATPase
(H-ATPase) of Saccharomyces cerevisiae is
required for the maintenance of intracellular pH and the large
electrochemical gradient that is necessary for the transport of many
nutrients (Goffeau and Slayman, 1981; Bowman and Bowman, 1986; Slayman,
1986; Goffeau and Dufour, 1988; Serrano, 1988). Yeast
H
-ATPase is structurally and functionally analogous to
the mammalian cation-translocating P-type ATPases including
Na
/K
-,
H
/K
-, and Ca
-ATPase
(Shull et al., 1985; MacLennan et al., 1986; Brandl et al., 1986). A comparison of the sequence of these enzymes
has shown extensive homology that includes most functional domains
(Serrano et al., 1986; Serrano, 1988). Like other P-type
ATPases, the yeast plasma membrane H
-ATPase has a
catalytic subunit of
100 kDa, hydrolyzes ATP via a transient
aspartylphosphate intermediate, and is inhibited by low concentrations
of vanadate. The H
-ATPase is a major cell protein,
representing about 15% of the plasma membrane protein and 0.3% of total
yeast protein (Serrano, 1991; Serrano, 1988; Bowman et al.,
1981). Genetic studies have shown that the PMA1 gene encoding
the H
-ATPase is essential (Serrano et al.,
1986).
The abundance of the yeast H-ATPase in the
plasma membrane appears to be tightly regulated, as over-expression of
the PMA1 gene on multicopy plasmids in yeast yields no
significant increase in the amount of PMA1 protein (Pma1) in
the plasma membrane (Eraso et al., 1987). This regulation does
not appear to occur at the transcriptional level but must occur at any
one of a number of steps during its proper entry into and progress
through the entire secretory pathway or in its maintenance in the
plasma membrane.
Many membrane proteins exist as part of
homo-oligomeric or hetero-oligomeric structures. Many of the animal
P-type ATPases form an active holoenzyme containing two subunits, a
100-kDa
subunit and a smaller
40-60-kDa
glycoprotein
subunit (Lingrel et al., 1988). The
subunit is considered the catalytic subunit while the
subunit
appears to be involved with the folding and maturation of the
subunit (Geering et al., 1987). The association of the
subunit with an appropriate
protein is required for exit from
endoplasmic reticulum (Jaunin et al., 1992) and subsequent
transport to the plasma membrane (Noguchi et al., 1987;
Takeyasu and Kawakami, 1989). For example, functional expression of
Na
/K
-ATPase in various expression
systems such as Xenopus oocytes or stably transfected cell
lines requires the co-expression of a compatible endogenous or
exogenous
subunit (Noguchi et al., 1987; Takeyasu et
al., 1988; Lemas et al., 1992). The requirement for the
expression of both
and
subunits is also seen when the
mammalian Na
/K
-ATPase genes are
expressed in yeast (Horowitz et al., 1990). In the case of the
Na
/K
-ATPase, the
subunit
appears to play an important role in catalysis and in interactions with
extracellular inhibitors (Geering, 1990). A
subunit is not
required for the catalytic activity of the Ca
-ATPases
(Wuytack and Racemackers, 1992). The yeast H
-ATPase
does not appear to require a
-like protein for its maturation,
transport, or functional expression on the plasma membrane. Although
there is a 115-kDa glycoprotein tightly associated with the
H
-ATPase during the purification (Vai et al.,
1988), the activity of the enzyme is normal in a mutant strain lacking
the glycoprotein (Serrano et al., 1991).
Previous research
has demonstrated that the yeast plasma membrane
H-ATPase activity is regulated in response to growth
conditions, increasing severalfold during glucose metabolism (Serrano,
1983) and in an acid medium (Eraso et al., 1987). This
regulation is likely to occur by one of several mechanisms of
post-translational modification. Protein phosphorylation, caused by
several protein kinases, has been demonstrated both for the
Ca
-ATPase (Caroni and Carafoli, 1981) and for the
yeast H
-ATPase (Chang and Slayman, 1991). In addition,
the C-terminal domain of the mammalian Ca
-ATPase can
interact with calmodulin to regulate its activity (Carafoli, 1992).
Allosteric regulation of the plant H
-ATPase enzyme by
a synthetic C-terminal peptide has also been reported (Palmgren et
al., 1991). Both mutation or deletion of the analogous C terminus
of the yeast H
-ATPase leads to the loss of glucose
activation (Portillo et al., 1989). Whether such regulation in
yeast depends on another interacting protein is unknown.
In this
paper, we describe the identification and characterization of MOP2, a gene that is essential in some strains and nearly so
in others. MOP2 protein behaves as a plasma
membrane-associated protein. Mutations in MOP2 reduce
dramatically the activity and abundance of Pma1 in the plasma membrane
without affecting the abundance of other prominent plasma membrane
proteins. Over-expression of MOP2 can rescue the inhibitory
effect on cell growth caused by high expression of PMA1. Surprisingly, MOP2 has recently been identified in two
very different searches of new yeast mutants. Holtzman et al. (1993) have identified mutations in the same gene as SLA2, synthetic lethal mutation in combination with a deletion
of an actin binding protein. At the same time, Raths et
al.(1993) have identified the same gene as END4,
essential for endocytosis. ()These results suggest that MOP2 (SLA2) plays a central role in the maintenance
and distribution of integral membrane proteins, including Pma1.
A haploid-viable, temperature-sensitive derivative of a mop2::URA3 deletion was obtained by transforming diploid DDY288 (Holtzman et al., 1993) with the URA3-marked disruption of MOP2 (SLA2) in plasmid pSN101, described below.
The media used in these experiments have been previously described (Perlin et al., 1989). Cells were grown in YEPD (1% (w/v) yeast extract, 2% (w/v) Bacto-peptone, 2% (w/v) dextrose, pH 5.5) or standard defined media lacking uracil or leucine. The ability of cells to grow under conditions of low external pH was tested in YEPD medium adjusted to pH 3.0 with HCl. Hygromycin B resistance was scored using YEPD containing 300 or 500 µg/ml hygromycin B. Cycloheximide hypersensitivity was scored using YEPD containing 0.5 µg/ml cycloheximide.
Figure 1:
Cloning and analysis of MOP2. A, deletion analysis of the MOP2-containing DNA
fragment from a genomic library. Both the 4.6-kb BamHI-XbaI and the 3.3-kb BamHI-PflMI fragment can complement all mop2 phenotypes. DNA sequence reveals that only the BamHI-XbaI fragment contains the intact MOP2 open reading frame. Restriction endonuclease sites are labeled as
follows: E, BstEII; B, BamHI; Bg, BglII; P, PvuII; Pf,
PflMI; X, XbalI. B, replacement of MOP2 open reading frame with URA3 gene in diploid SN189.
Deletion of MOP2 caused cell lethality, as evidenced by the
recovery of only 2 viable (Ura) spores per tetrad. C, location of a putative membrane-spanning domain and point
of insertion of the c-Myc epitope.
Plasmid pSN145 is a LEU2-marked high copy plasmid in which the BamHI-XbaI fragment of plasmid pSN117 carrying the epitope-tagged MOP2 gene was inserted in place of the BamHI-NheI fragment of plasmid YEp21.
Plasmid pNS146 is a derivative of pNS110 containing a 1.1-kb URA3 fragment inserted at an NruI site in the 3`-non-coding region of the epitope-tagged MOP2 gene.
ATPase activity was assayed as described (Perlin et al., 1989). Total protein concentration was determined by
the Lowry method. Abundance of H-ATPase in purified
plasma membrane preparations was examined by SDS-PAGE electrophoresis.
Samples were solubilized in SDS-PAGE loading buffer and incubated at 37
°C for 10 min before loading on a 10% SDS-PAGE gel. Proteins were
visualized with Coomassie Blue or by Western blotting. For Western blot
analysis, proteins were transferred to nitrocellulose membranes.
Transfer buffer was 25 mM Tris, 192 mM glycine, 10%
methanol, 0.03% SDS, pH 8.4. Transfer was carried out for 1.5 h at a
constant current of 275 mA. Protein on immunoblots was detected using a
chemiluminescence detection system according to the protocol provided
by the manufacturer (Amersham Corp.).
The effect of mop2 on
the abundance of Pma1 was determined by comparing a wild type (SN197)
and a mop2-2 mutant (SN53) strain. Cells were grown at
25 °C in YEPD media as described above. When cells reached an A = 1, the cultures were divided into
three flasks. Growth was continued in one flask at 25 °C for 4 h
and for either 2 or 4 h at 37 °C in the other two flasks. Cells
were harvested, and plasma membranes were prepared.
Solubilization
of Mop2 from plasma membranes was carried out by treating the purified
plasma membrane with chemical reagents. 100 µg of purified plasma
membranes from SN330 (transformant of Y55-296 with pSN117) were
resuspended in 200 µl of one of the following solubilizing
reagents: 1% Triton X-100, 1% SDS, 1% Zwittergent-14 (Boehringer
Mannheim), 2 M urea, or 0.1 M NaCO
, pH 11. Samples were incubated on ice
for 1 h. As a control, plasma membrane was incubated with membrane
buffer (10 mM Tris-Cl, pH 7.5, 1 mM EDTA, 10%
glycerol) under the same conditions. Each mixture was subjected to
sedimentation for 1 h at 100,000 rpm in a table top ultracentrifuge.
Each pellet was resuspended in 200 µl of 10 mM Tris
buffer, pH 7.5. Equivalent amounts of the high speed supernatant and
the solubilized pellet were then analyzed by Western blot, probed with
the 9E10 anti c-Myc monoclonal antibody.
Figure 2: Phenotypes of pma1-114, mop2-1, mop2-2, SH89, and the wild type strain double mutant pma1-114 mop2-1. pma1-114 (Perlin et al., 1989) is resistant to hygromycin B at a concentration of 300 µg/ml but sensitive to hygromycin B at 500 µg/ml and sensitive to YEPD + 0.2 M acetate, pH 4.8. mop2-1 is hypersensitive to cycloheximide (YEPD + 0.5 µg/ml) and sensitive to YEPD + 0.2 M acetate, pH 5.0. mop2-2 has the same phenotype as mop2-1 but is also temperature sensitive (no growth at 37 °C). The phenotypes of higher concentration hygromycin B resistance, low pH sensitivity, and high osmotic stress sensitivity are only seen for the double mutant pma1-114 mop2-1.
Three additional mop2 alleles (mop2-2, mop2-3, and mop2-4) were subsequently isolated by their hyper-hygromycin B resistance of pma1-114. Both mop2-2 and mop2-4 were also found to be temperature sensitive for growth at 37 °C.
Strains carrying mutations in both MOP2 and PMA1 have several interesting phenotypes that are absent in the mop2 and pma1 mutants. Although some severe pma1 alleles are unable to grow at pH 3.0 and are sensitive to osmotic stress (McCusker et al., 1987), pma1-114 is resistant to both of these conditions, although it is hygromycin B resistant and sensitive to acid loading (YEPD + 0.2 M acetate, pH 4.8). mop2 pma1-114 strains are sensitive to low external pH (YEPD, pH 3.0) and high osmotic medium (YEPD plus 2 M glycerol) in addition to showing higher resistance to hygromycin B (Fig. 2). This proved to be true of both mop2-1 and mop2-3 and for the temperature-sensitive mop2-2 and mop2-4 alleles grown at their permissive temperature of 25 °C.
Figure 3:
Abundance of H-ATPase in
wild type (SN197) and mop2-2 (SN53) strains. A,
plasma membranes derived from SN197 and SN53 were analyzed by SDS-gel
electrophoresis on a 10% gel (40 µg/lane). Membranes were prepared
from both SN197 and SN53 cells grown at 25 °C (the permissive
temperature for mop2-2) and 37 °C (non-permissive
temperature) as described under ``Experimental Procedures.'' Lane1, wild type (25 °C); lane2, wild type (37 °C, 2.0 h); lane3, wild type (37 °C, 4.0 h); lane4, mop2-2 (25 °C); lane5, mop2-2 (37 °C, 2.0 h); lane6, mop2-2 (37 °C, 4.0 h). B,
immunoblot of the same plasma membranes as in A. 5 µg/lane
were loaded on a 10% mini SDS-PAGE gel and transferred to
nitrocellulose membrane. Blot was probed with anti-Pma1 monoclonal
antibody.
A further experiment was done by replacing the PMA1 promoter with the galactose-inducible GAL1 promoter (Cid et al., 1987) to test whether the effect of mop2 on
the abundance of the Pma1 could be suppressed by placing the PMA1 gene under the control of another promoter. Strain SN225 was
constructed by crossing the mop2-2 strain, SN234 (MAT ho::LEU2 ura3-1 leu2-1 arg4-1
mop2-2 GAL3), with a MAT
spore of strain
SH132, which contains PMA1 under galactose-inducible promoter. The
correct haploid meiotic segregants, confirmed by both Southern blot and
scoring for the mop2-2 spore colonies that grow on
galactose, were used for measurement of Pma1 abundance in plasma
membranes. SDS protein gels of the purified plasma membrane showed that
abundance of Pma1 was still reduced by mop2 to the same extent
as the cells with the normal PMA1 promoter (data not shown).
These experiments demonstrated that MOP2 does not act at the
level of PMA1 transcription.
MOP2 was
mapped to chromosome XIV by probing a Southern blot of a yeast
chromosome separating gel (data not shown) using P-labeled
cloned MOP2-containing DNA fragment. Tetrad analysis was then
carried out to establish that MOP2 lies approximately 7
centimorgans proximal to RAD50 on the left arm of chromosome XIV (data not shown). There are no other known genes in this
vicinity (Mortimer et al., 1992).
In the clone from the YEp24 genomic bank, MOP2 was contained in a 10-kb insert. To narrow down the fragment that encodes MOP2, several deletions of the MOP2 clone were made and tested for their functional complementation (Fig. 1A). The smallest fragment that complemented a mop2 mutant was the 3.47-kb BamHI-PflMI fragment. However, DNA sequencing of MOP2 shows that the Mop2 open reading frame extends 63 amino acids beyond the PflMI site (see below). This suggests that at least the last 63 amino acids of C terminus are not essential for Mop2 function. Moreover, an insertion of c-Myc epitope at the PflMI site of MOP2 did not affect the function of Mop2 (see below).
A null mutation of MOP2 was created
by removal of almost the entire protein coding sequence and replacement
of this segment with the selectable marker URA3 (see
``Experimental Procedures''). This construct, mop2::URA3, was transformed into a derivative of diploid
strain, Y55 (HO ura3-1). Stable Ura
transformants were selected, and the deletion of MOP2 was confirmed by Southern blot analysis (data not shown). A
Ura
diploid was sporulated and dissected. Only two
viable spores were obtained in each tetrad, all of which were
Ura
, indicating that MOP2 encodes an
essential protein for cell growth. This result is in contrast to that
of Holtzman et al.(1993), who found that a virtually identical
deletion of the same gene in a different strain yielded viable haploids
that were temperature sensitive for growth. It was likely that our
strains, based on strain Y55 (McCusker and Haber, 1988), harbor a
genetic difference from those used by Holtzman et al.(1993).
To confirm this genetic difference, we transformed the same mop2::URA3 deletion construct we had introduced into our
strain Y55 into strain DDY228 used by Holtzman et al.(1993).
Tetrads of Ura
derivatives of DDY228 were sporulated
and dissected at 25 °C. After 3 days, there were only two visible
(Ura
) segregants. However, in contrast with strain
Y55, by 5 days two Ura
colonies become visible. These
very slow growing colonies proved to be unable to grow at 37 °C, in
agreement with Holtzman et al.(1993). Thus, there appears to
be a genetic difference between the two strains so that deletions of MOP2 (SLA2) are lethal in one strain but viable
(though very debilitated) in another.
To determine the nature of the membrane association of Mop2, the purified plasma membranes from strain SN330, containing a high copy plasmid with MOP2::c-Myc, were treated with a variety of reagents and resedimented at 100,000 rpm. Equivalent portions of the resulting supernatant were analyzed by immunoblotting (see ``Experimental Procedures''). Mop2 protein was found exclusively in the membrane fraction. No immunoreactivity was found in the high speed supernatant fraction (Fig. 4B, lane2). The results showed that Mop2 is partially extracted by the reagents known to extract peripherally associated membrane proteins and is efficiently extracted by SDS and Zwittergent-14 known to solubilize integral membrane proteins (Fig. 4B). Triton X-100 is less efficient compared with the other two detergents. Taken together, these data indicate that Mop2 itself is a plasma membrane-associated protein.
Figure 4: Mop2 is a membrane-associated protein. A, immunoreactivity of untagged Mop2 (lane1) and tagged Mop2::c-Myc (lane2) Western blot was probed with anti-c-Myc mAb 9E10. B, solubilization of Mop2 from the purified plasma membrane. Purified plasma membrane was isolated from SN330 carrying pSN117 (see ``Experimental Procedures''). 10 µl of high speed pellet (P) and supernatant (S) fractions were analyzed by Western blot. The nitrocellulose membrane was probed with the 9E10 anti-c-Myc mAb.
In some preparations we find two bands, approximately 92 and 108 kDa, that were highly stained with anti c-Myc monoclonal antibody 9E10 (Fig. 4A). The size of 108 kDa is consistent with the predicted molecular size from the MOP2 sequence. The 92-kDa band is most likely a proteolytic digestion product of Mop2. Another protein of approximately 100 kDa is found both in cells expressing MOP2::c-Myc and those expressing an untagged Mop2 protein. This second protein is most likely Pma1, as shown by immunoprecipitation with anti-Pma1 antibodies (data not shown). Immunoreactivity of anti-c-Myc antibody with this protein has been seen previously (Kuchler et al., 1993). Immunoprecipitation of Pma1 does not precipitate the 108-kDa Mop2::c-Myc protein (data not shown). The strength of anti-c-Myc immunoreactivity with Pma1 is strain dependent, as it is less evident in strains provided by A. Goffeau (Supply et al., 1993) (see below).
Further evidence of the plasma membrane location of Mop2 came from immunofluorescence staining by monoclonal anti c-Myc antibody of the cells carrying the plasmid containing epitope-tagged Mop2. The cells showed bright staining of the cell periphery (Fig. 5), as do antibodies against Pma1 (Harris, et al., 1994) and other plasma membrane proteins (Ljungdahl et al., 1992). Despite the fact that anti-c-Myc antibody reacts with Pma1 on Western blots, it does not exhibit any significant indirect immunofluorescence in cells not expressing Mop2::c-Myc (Fig. 5). We conclude that Mop2 is located at the plasma membrane.
Figure 5: Localization of Mop2 by indirect immunofluorescence. Strain YPS3-2A carrying pSN145 was grown in standard defined complete medium lacking leucine for selection of plasmid. Cells carrying the MOP2::c-Myc gene on a high copy plasmid pSN145 (A) and the same strain carrying a similar plasmid but lacking the MOP2 gene, YEp21 (B), were examined by indirect immunofluorescence using an anti-c Myc mAb antibody (toppanels). Lowerpanels, cells viewed by phase contrast and stained with 4,6-diamidino-2-phenylindole.
Figure 6:
Growth curve of both the wild type
(Y55-296) and mop2-2 mutant (SN53) strains
carrying one extra copy or high copy plasmid with PMA1, MOP2, or both PMA1 and MOP2. All of the
strains were grown at 25 °C with continuous shaking. Measurement of
cell growth was done by measuring absorbance at 600 nm. A,
growth curve of wild type cells with high copy PMA1 or MOP2 or both (, + YEp24;
, + high copy
plasmid containing PMA1;
, + high copy plasmid
containing MOP2 (pSN117); [tirf], + both high
copy plasmids containing MOP2 and PMA1). B,
growth curve of wild type cells and mop2-2 mutant cells
with different copies of PMA1 (
, wild type + YEp24;
, mop2-2 + YEp24;
, mop2-2 + high copy plasmid containing PMA1;
, mop2-2 + single copy (centromeric) plasmid
containing PMA1 (pSN141).
We also measured the abundance of Pma1 protein in these cells. The SDS-PAGE gel and Western blot depicted in Fig. 7show that the level of Pma1 in the plasma membrane does not change in cells containing different numbers of PMA1 genes. Moreover, the addition of a high copy MOP2 plasmid has no effect on the amount of Pma1 observed. We conclude that the relief of toxicity of over-expressing PMA1 by over-expressing MOP2 may involve a change in the way excess Pma1 protein is transported or turned over and that this requires the participation of the Mop2 protein.
Figure 7: Effect of over-expressing PMA1 and MOP2 on the abundance of Pma1 protein. A, 30 µg of purified plasma membranes were analyzed by SDS-PAGE on a 10% mini gel. Lane1, wild type; lane2, single copy PMA1 (centromeric plasmid); lane3, high copy PMA1 plasmid; lane4, both high copy PMA1 and high copy MOP2 plasmids; lane5, high copy MOP2 plasmid; lane6, molecular weight markers. B, immunoblot of the same plasma membrane samples as in A. Nitrocellulose membrane was probed with anti-Pma1 mAb. In some lanes, a band consistent with being a Pma1 dimer is seen.
Interestingly, wild type and mop2 mutant cells exhibited different sensitivity to the over-expression of PMA1. In mop2-2 cells grown at permissive temperature, a single extra copy of the PMA1 on a centromere plasmid was as deleterious to the rate of cell growth as with the high copy PMA1 plasmid, while growth is inhibited only by high copy PMA1 in wild type cells (Fig. 6B). These results suggest that MOP2 interacts with PMA1 protein and apparently regulates the transport accumulation or turnover of Pma1 protein.
MOP2 was identified by mutations that enhance the
phenotypes of pma1 mutants. This enhancement appears to be
caused by markedly reducing the abundance of the mutant Pma1 protein in the plasma membrane. All of the evidence suggests that MOP2 encodes an essential (in our strains) plasma
membrane-associated protein that is required for the proper
accumulation of the H-ATPase in the plasma membrane.
This effect on Pma1 protein is not simply a general change in membrane
protein abundance of large (>30 kDa) plasma membrane proteins.
Further evidence of a direct relationship between the abundance of
functional Mop2 and the abundance of Pma1 protein comes from our
finding that the detrimental effects on cell growth by over-expression
of PMA1 can also be rescued by over-expression of MOP2. Conversely, a mop2 mutant strain becomes
hypersensitive to an increase in PMA1 copy number. In this
discussion, we try to understand what the primary role of Mop2 might
be, especially in view of the finding that MOP2 (SLA2) has recently been identified by two other mutant
screens based on very different aspects of cell biology (Holtzman et al., 1993; Raths et al., 1993). (
)
Reduction of Pma1 on the plasma membrane could, in principle, be due to any defect in the transport or subsequent degradation pathway caused by the mop2 mutation. Based on both biochemical characterization and immuno-electron microscopic study of the transport vesicles (Holcomb et al., 1988; Brada and Schekman, 1988), it has been shown that Pma1 is transported in the same secretory vesicles as other secretory proteins, including invertase and acid phosphatase. Defects in several SEC genes block the transport of Pma1 protein to the plasma membrane and result in the accumulation of secretory vesicles (Nakamoto et al., 1991; Chang et al., 1993). It does not appear that mop2 blocks Pma1 protein transport in the same way, since we do not see any accumulation of Pma1 protein in the endoplasmic reticulum or any other intracellular locations when we examine mop2-2 strains by immunofluorescence staining at either their permissive (but still mutant) or non-permissive temperature (data not shown). We know such accumulation can be seen by our immunofluorescence methods, as we find very significant accumulation of Pma1 in cells expressing dominant lethal pma1 mutations (Harris et al., 1994). Also, no change in the abundance of Pma1 was observed in cells that overexpress PMA1. This suggests that the regulation of Pma1 abundance might occur at the level of the plasma membrane.
The fact that MOP2 is a plasma
membrane-associated protein is compatible with several suggestions
about the way it affects the abundance of Pma1. One possibility is that
Mop2 functions as a necessary and specific transport protein in the
secretion of H-ATPase, similar to the SHR3 protein identified for amino acid permeases (Ljungdahl et
al., 1992). Mop2 differs from classic chaperone proteins in that
it does not remain in the endoplasmic reticulum or Golgi apparatus but
instead is itself found at the plasma membrane.
Alternatively, Mop2
might act similarly to the subunit of some mammalian P-type
ATPases to form a heterodimer with the
subunit that is required
for its transport to the plasma membrane. There has been no evidence
for such a transport subunit for H
-ATPase in yeast,
but several recent findings are consistent with the idea that such a
-like protein exists in yeast and is required for
H
-ATPase transport to the plasma membrane. For
example, when the Arabidopsis plasma membrane
H
-ATPase is expressed in S. cerevisiae, the
plant enzyme is abundant and is fully functional in terms of ATP
hydrolysis, formation of a phosphorylated intermediate, and ability to
pump protons in reconstituted vesicles (Villalba et al.,
1992). However, the plant plasma membrane H
-ATPase is
not transported to the yeast plasma membrane but remains trapped in the
endoplasmic reticulum. Since the accumulated plant
H
-ATPase is fully functional in vitro, the
presence of plant H
-ATPase in the endoplasmic
reticulum seems unlikely to be due to the improper folding of the
enzyme. The plant and yeast H
-ATPase share extensive
homology in their membrane structure and function; nevertheless, the
plant homologue is unable to traverse the secretory apparatus, possibly
because it cannot interact properly with a Pma1-specific transport
protein. One argument against Mop2 being a
subunit is that its
abundance in the plasma membrane can be significantly increased in the
plasma membrane by over-expressing MOP2, but at the same time
Pma1 does not hyperaccumulate.
Another possibility is that Mop2
might be needed to combine with and stabilize H-ATPase
once it has reached the plasma membrane. It is conceivable that mop2 mutations might accelerate the endocytosis and turnover
of Pma1 protein on the plasma membrane. Pma1 is normally a
long-lived protein with a half-life of 30 h (Benito et al.,
1991). Its half-life in mop2 mutants has not been measured.
At the same time, sla2 mutations of the same gene were found as synthetic lethal mutations in a strain already deleted for the actin binding protein, ABF1 (Holtzman et al., 1993). sla2 mutations appear to profoundly affect the actin-based cytoskeleton of the cell, as evidenced by a disruption of the polarized distribution of actin cortical granules. Moreover, temperature-sensitive alleles of sla2 frequently have multinucleate cells with altered morphology (a phenotype we do not see in our cells). The fact that MOP2 (SLA2) shares apparently significant homology with the cytoskeleton-associated protein, talin, in mammalian cells (Reed et al., 1990) provides a satisfying analogy, although we have shown that a deletion of the last 63 amino acids including about of this homology has no effect on the activity of MOP2 (SLA2).
Part of the difficulty in understanding how mop2 mutant phenotypes relate to sla2 mutant phenotypes comes from the fact that the strains used to analyze the two sets of mutations are quite different, and, in fact, a deletion of the MOP2 (SLA2) gene in our strains is lethal at any temperature, while an identical deletion in the strains used to isolate sla2 leads only to very delayed germination, slow growth, and temperature-sensitive conditional lethality. The genetic difference in our strains has not been elucidated.
One way to understand the effect of sla2 mutants on Pma1 abundance comes from the observation in mammalian
cells of another member of the cation ATPase superfamily. The mammalian
Na/K
-ATPase is found asymmetrically
distributed in polarized epithelia and that this distribution involves
associations with a number of membrane cytoskeleton proteins including
ankyrin and fodrin (Marrs et al., 1993; Hammerton et
al., 1991; Nelson and Veshnock, 1987). In this view, an alteration
in the distribution and disruption of the yeast cytoskeleton would
alter sites where Pma1 would normally be located in the cell membrane.
Given previous evidence that new secretory vesicle insertions into the
plasma membrane occur predominantly in the growing yeast cell bud
(Holcomb et al., 1988; Brada and Schekman, 1988), it is
understandable that a mutation that altered this normal polarity would
reduce the abundance of Pma1 in the plasma membrane. Similarly, defects
in the cytoskeleton might prevent normal endocytosis.
Although we can demonstrate an interaction between Mop2 and Pma1 in several ways, it is not yet clear that all of the properties of mop2 mutants can be explained by its role in regulating the abundance of Pma1. mop2 also exhibits hypersensitivity to cycloheximide. This might imply that mop2 might also affect other membrane properties. Cycloheximide hypersensitivity cannot simply be explained as the lowering of membrane potential by affecting Pma1 abundance because pma1 mutants, even those such as pma1-155 that decrease the abundance of wild type protein, do not share this phenotype (Perlin et al., 1989).
The yeast Pma1 is a major plasma membrane protein
accounting for 15% of total plasma membrane protein. The abundance of
Pma1 is quite constant during the changes in growth conditions or after
increasing PMA1 gene dosage; in fact, over-production of the PMA1 slows yeast cell growth (Eraso et al., 1987).
There may be structural constraints against increasing the amount of
H-ATPase without affecting the integrity of the
membrane. Another possibility is that H
-ATPase is a
major ATP consumer, and its over-production may compromise the energy
charge of the cell (Serrano, 1980). Therefore, it is possible the yeast
cells need to regulate the abundance of H
-ATPase in
the plasma membrane. Mop2 appears to be one of the components that
controls H
-ATPase abundance.
Note Added in Proof-Recently, we have found that under some physiological conditions, overexpression of PMA1 can suppress the temperature sensitivity of mop2-2 (M. Hincapie and J. E. Haber, unpublished results).