From the Department of Biology, Tokyo Gakugei
University, Tokyo 184-8501, the § Department of Life
Sciences, Graduate School of Arts and Sciences, The University of
Tokyo, Tokyo 153-8902, and ¶ CREST, Japan Science and Technology
Corporation, Saitama 332-0012, Japan
Received for publication, July 12, 2002, and in revised form, December 5, 2002
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ABSTRACT |
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The Saccharomyces cerevisiae
MID1 gene product, Mid1, is composed of 548 amino acid residues,
has four relatively hydrophobic segments named H1-H4, and functions as
a Ca2+-permeable, stretch-activated channel when expressed
in mammalian cells. In some conditions Mid1 cooperates with Cch1, a
yeast homolog of the The MID1 gene product, Mid1, is an
N-glycosylated, integral membrane protein required for
viability of differentiated yeast cells and Ca2+ influx
induced by the mating pheromone Genetic and cell biological studies have revealed that Mid1
coimmunoprecipitates and works with Cch1, a yeast homolog of the pore-forming Although the physiological roles of Mid1 have been elucidated as
described above, the structure-function relationship of this protein
remains unclear. The Mid1 polypeptide is composed of 548 amino acid
residues and could form a homotetramer (see supplemental material in
Ref. 2), having four hydrophobic segments, H1-H4 (1) (see Fig.
1). It is uncertain whether these
segments are transmembrane domains. H1 is probably a signal peptide
(1). Computational analysis with the TMpred program (available at
www.ch.embnet.org/software/TMPRED_form.html) suggests that H3 and H4
are possible transmembrane helices. The hydrophobic profile of H3 is
similar to that of the pore-forming regions of several cation channels
(see supplemental material in Ref. 2). H4 is partially homologous to
the S3/H3 membrane-spanning domain of several ion channels (1).
Protease protection experiments on intact cells have revealed that Mid1
is present in the plasma membrane and that its C-terminal region is in
the cytoplasm (1). The C-terminal, cytoplasmic region downstream from
H4, including the cysteine-rich regions, is essential for Mid1 function
(22).
1 subunit of mammalian voltage-gated channels.
To identify the important regions or amino acid residues necessary for
Mid1 function, we employed in vitro site-directed
mutagenesis on H3 and H4 of Mid1 and expressed the resulting mutant
genes in a mid1 null mutant to examine whether the mutant
gene products are functional or not in vivo. Mutant Mid1
proteins lacking the whole H3 or H4 segment, H3De or H4De, did not
complement the lethality and low Ca2+ accumulation activity
of the mid1 mutant, although their localization and
contents appeared to be normal, indicating that H3 and H4 are required
for Mid1 function itself. Single amino acid exchange experiments on
individual amino acid residues of H3 and H4 showed that 10 of 20 residues in H3 and 14 of 23 residues in H4 were important for the
normal function of Mid1. In particular, we found four severe
loss-of-function mutations, D341E, F356S, C373D, and C373R, and two
interesting mutations leading to a high level of Ca2+
accumulation with a slightly low complementing activity, G342A and
Y355A. The importance of these amino acid residues will be discussed.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-factor (1). Cells lacking the
MID1 gene die because of limited Ca2+ influx
when incubated with
-factor. This phenotype is called the
mid phenotype for the mating
pheromone-induced death phenotype. Electrophysiological and cell biological studies on Chinese hamster ovary cells, Balb/c 3T3 cells, and COS-7 cells expressing Mid1 have
revealed that it functions as a Ca2+-permeable
stretch-activated channel (2, 3). The mating pheromone leads their
target cells to differentiate into cells (so-called "shmoos")
having a mating projection at which the plasma membrane and the cell
wall are degraded and regenerated (4, 5). Concomitantly with the
formation of the mating projection, Mid1-dependent
Ca2+ influx is stimulated (1, 6, 7). Thus, Mid1 might
function in sensing membrane stretch and generating Ca2+
signals during the mating process. Although stretch-activated channels
or mechanosensitive channels are known to play a critical role in touch
sensation, hearing, balance, detecting gravity, and sensing osmotic
changes, little is known about their molecular structures and
biochemical properties.
1 subunit of mammalian voltage-gated Ca2+
channels (8-10). The voltage-gated Ca2+ channels are
heteromultimeric proteins consisting of
1, cytoplasmic
, and
non-pore-forming transmembrane
2
and
subunits (11). These
non-pore-forming subunits dramatically influence the properties and
surface expression of the channels (12-16). Although the channel activity of Cch1 has not yet been revealed experimentally and Mid1 has
no homology to the auxiliary subunits, Mid1 has been shown to cooperate
with Cch1 in mating pheromone-induced Ca2+ uptake (8, 9,
17), store-operated or capacitative Ca2+ entry (10),
endoplasmic reticulum stress-induced Ca2+ uptake (18), and
a hyperosmotic stress-induced increase in cytosolic Ca2+
(19). On the other hand, it has been shown recently that Mid1, but not
Cch1, is required for an antiarrhythmic drug amiodarone-induced increase in cytosolic Ca2+ mainly caused by
Ca2+ influx (20). Mid1 is also involved in a
hexose-induced, transient elevation of cytosolic Ca2+ (21).
Therefore, Mid1 might function as an stretch-activated channel alone in
some cellular situations and as a regulator of another channel composed
of Cch1 in other cellular situations. This speculation remains to be
proven at the molecular level.
View larger version (17K):
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Fig. 1.
Schematic diagram and hydropathy profile of
Mid1. Mid1 has four hydrophobic regions, H1-H4, 16 putative
N-glycosylation sites ( ), and 15 cysteine residues (
).
Position numbers of amino acid residues are indicated at the
top of the figure.
In this study, to explore the structure-function relationship of H3 and
H4, we mutated each one of all 20 and 23 amino acid residues in H3 and
H4, respectively, and tested the effects of these single point mutants
on Mid1 function by examining the ability of the mutant proteins to
complement the mid phenotype and low Ca2+
accumulation of the mid1 mutant. From these analyses, we
identified several key amino acid residues and their clusters that
potentially contribute to Ca2+ permeability.
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EXPERIMENTAL PROCEDURES |
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Strains and Culture Conditions--
The yeast strains used in
this study are listed in Table I. Rich
media and a synthetic medium, SD, were prepared as described previously
(23). Yeast nitrogen base was prepared according to the formula given
in the Difco manual (24). Because SD medium contains 680.2 µM CaCl2 and 0.8 µM calcium
pantothenate, in the Ca2+-deficient medium SDCa,
CaCl2 was omitted, and calcium pantothenate was replaced
with sodium pantothenate. SD.Ca100 medium was prepared by adding 100 µM CaCl2 to SD
Ca medium. Synthetic
presporulation medium contained 6.7 g of yeast nitrogen base and
10 g of potassium acetate/liter. These media were supplemented
with the appropriate nutrients as described previously (23). The
sporulation medium contained 10 g of potassium acetate/liter.
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Escherichia coli strain used was XL1-Blue (Stratagene).
Competent cells were prepared according to the methods described
previously (25). Luria-Bertani medium and terrific broth were prepared as described previously (26). When required, ampicillin was added at 50 µg ml1.
Site-directed Mutagenesis-- To construct plasmids with the MID1 gene having deletion mutations or single mutations, site-directed mutagenesis was employed. The mutagenic primers were ordered from Invitrogen, and their DNA sequences are listed in Table II (supplemental data available at JBC on-line). PCR was performed with Pfu polymerase (Stratagene) and YCpMID1-23 or YEpMID1-GFP1 (Table I) as a PCR template. The PCR Thermal Cycler MP (Takara) was used for PCR. The low copy plasmid YCpMID1-23 and its derivatives were used for measuring the viability of cells and Ca2+ accumulation and immunoblot analysis; the multicopy plasmid YEpMID1-GFP and its derivatives were used for detecting Mid1-GFP by confocal microscopy and immunoblot analysis. Successful mutagenesis was confirmed by DNA sequencing using an ABI Prism Automated Sequencing Kit and an ABI 310 Automated Sequencer (Applera Co., Applied Biosystems).
Construction of a mid1 Null Mutant, mid1-5--
Any
mid1 null mutant producing no Mid1 polypeptide was not
constructed by inserting a marker gene near the initiation codon of the
MID1 gene because the 5'-coding region of the
MID1 gene is necessary for the expression of the adjacent,
essential gene RFC3 (1). To construct a null mutant of the
MID1 gene (mid1-
5) by
site-directed mutagenesis, EpMID1-
5 was prepared by introducing the
stop codon TAA at Ile2 of the Mid1 protein coded for the
plasmid EpMID1-31-P-HIS3-2 (1) using a set of synthetic primers,
5'-GTATTATTTCTAAGGATGTAAGTGTGGC-3' and
5'-GCCACACTTACATCCTTAGAAATAATAC-3', where the
underline indicates the stop codon and its anticodon, respectively. The
resulting plasmid EpMID1-
5 was then used as a template to synthesize
a DNA fragment containing the mid1-
5 gene by
PCR using a set of synthetic primers, 5'-GATATAGTTTCGCTGCCATC-3' and
5'-CGTATCGTCCAATGGATGAATTCACATCAAGGATGAGTTACC-3'. The DNA fragment was
used to transform a wild-type strain, H207, to produce H311 bearing the
mid1-
5 mutation. Successful construction of
the mid1-
5 allele was confirmed by PCR, DNA
sequencing, and tetrad analysis. Immunoblot analysis confirmed further
that the Mid1 protein was not produced from the
mid1-
5 allele at all.
Transformation of Saccharomyces cerevisiae Cells-- The S. cerevisiae strain H311 was transformed by various plasmids whose selection marker was LEU2 (Table I) according to the method described previously (27), with minor modifications. The transformants were selected on agar plates containing SD medium supplemented with 20 µg/ml uracil and 20 µg/ml tryptophan at 30 °C for 3 days.
Activity of the Mutant Mid1 Proteins--
To examine the
activity of the mutant Mid1 proteins, the viability and
Ca2+ accumulation of cells producing the proteins were
measured by the methods described previously (1). The mating pheromone -factor was ordered from the Center for Analytical Instruments, National Institute for Basic Biology, Okazaki, Japan.
GFP Fluorescence Imaging-- The Mid1-GFP fusion proteins having mutations were expressed in the strain H311. Cells in the exponential phase in SD medium were harvested and placed on slide glasses covered with 1% poly-L-lysine hydrobromide. Subsequently, the slide glasses were sealed under a coverslip with nail polish. GFP images were observed using a confocal fluorescence microscope equipped with Nikon TE 300 (Nikon) in conjunction with Micro Radiance (Bio-Rad). We used Nikon Plan Apo 60x/1.40 Oil (Nikon) as an objective lens. Images were processed using Adobe Photoshop 5.0 (Adobe Systems).
Preparation of Cell Extracts and Immunoblot Analysis-- The methods of cell extract preparation were described previously (1) and used with slight modifications. An improved immunoblotting technique was employed (28). Cell extracts containing the Mid1 protein were applied on a 7.5% SDS-polyacrylamide gel and detected using rabbit polyclonal antibodies against the glutathione S-transferase-Mid1 (GST-Mid1) fusion protein at a 1:2,000 dilution (22). Enolase was detected using rabbit polyclonal antibodies against yeast enolase at a 1:5,000 dilution (29) and used for an internal marker for the amount of protein applied.
Statistical Analysis--
Statistical significance was
determined using an unpaired Student's t test, with a
maximum p value of < 0.05 required for significance.
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RESULTS |
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H3 Is Essential for Mid1 Function--
To determine whether H3 is
essential for Mid1 function, a mutant Mid1 protein lacking the whole H3
segment from Ile337 to Phe356, H3De, was
constructed by site-directed mutagenesis using the low copy plasmid
YCpMID1-23 (1) as a template and expressed in the
mid1-5 mutant. The mutant expressing H3De, the
H3De mutant, was incubated with
-factor for 8 h and examined
for the viability of the cells. The result shows that the H3De mutant
protein did not complement the mid phenotype of the
mid1-
5 mutant (Fig.
2A). Because the
mid1 mutations result in a low activity in mating pheromone-induced Ca2+ accumulation (1), we measured
Ca2+ accumulation in the H3De mutant exposed to
-factor
for 2 h. Ca2+ accumulation in the H3De mutant was low,
like that in the mid1-
5 mutant containing the
vector pRS315 (Fig. 2B). The result suggests that the H3
segment is required for Mid1 function.
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To identify important amino acid residues in H3, all 20 amino acid
residues in this segment were mutated individually by site-directed mutagenesis with the following rules. All of the nonpolar amino acids
were replaced by a polar amino acid, Ser, and the polar amino acids
were replaced by a small, nonpolar amino acid, Ala. Gly is classified
as a polar amino acid and was thus replaced by Ala. Because it has been
reported that the negatively charged amino acids Glu and Asp in the
transmembrane domain contribute to ion selectivity in voltage-gated
Ca2+ channels (30, 31) and ECaC (32, 33) and to interaction with channel blockers (34, 35), the remarkable Asp341 was
replaced by each of 7 amino acids, Glu, Lys, Arg, Thr, Asn, Ala, and
Leu, and the positively charged amino acid His353 was also
replaced by each of 3 amino acids, Asp, Arg, and Ala. The mutant genes
bearing these single amino acid substitutions were then introduced into
cells of the mid1-5 mutant, and the resulting
transformants were examined for viability and Ca2+
accumulation after exposure to
-factor. The results showed that the
most notable Mid1 mutant protein was D341E, which had almost completely
lost both the activity of complementing the mid phenotype and Ca2+ accumulation activity (Fig. 2). F356S also lost
the complementing activity completely (Fig. 2A) but still
had Ca2+ accumulation activity near the wild-type level
(Fig. 2B). G342A, I349S, V354S, and Y355A had slightly lower
complementing activities than the wild-type protein, but two of which,
G342A and Y355A, had greater Ca2+ accumulation activity
than the wild-type protein, whereas the remainder had normal
Ca2+ accumulation activity. In contrast, I337S, N339A, and
D341A had slightly greater complementing activity than the wild-type
protein, among which I337S had a significantly increased
Ca2+ accumulation activity and the remainder had normal
Ca2+ accumulation activity. The above observation that
G342A and Y355A had a significantly lower complementing activity than
the wild-type protein despite having a greater Ca2+
accumulation activity suggests at least two possibilities. One is that
overaccumulation of Ca2+ may lower the viability of cells.
The other is that Mid1, as a regulator, might activate Cch1 and
inactivate another factor responsible for cell viability.
It is possible that some of the above mutations are dominant negative
mutations rather than loss-of-function mutations. To test this
possibility, the low copy plasmid containing severe mutations, such as
H3De, D341E, or F356S, was introduced into wild-type cells, and the
resulting transformants were examined for viability 8 h after the
addition of -factor. The results showed that the viabilities of
these transformants were normal (data not shown), indicating that the
mutations tested are recessive and loss-of-function ones.
H4 Is Essential for Mid1 Function--
A mutant Mid1 protein
lacking the whole H4 segment from Leu366 to
Gly388, H4De, lost the activity of complementing the
mid phenotype as well as Ca2+ accumulation
activity as did the H3De protein under the same conditions (Fig.
3). To identify important amino acid
residues in H4, all 23 amino acid residues in the segment were mutated individually by site-directed mutagenesis with the same rules as those
in H3. Cys373 was replaced by each of 3 amino acids, Asp,
Arg, and Ala, because Cys is a highly reactive amino acid residue. The
results showed that the most marked mutant proteins were C373D and
C373R (Fig. 3). The two proteins did not have either the complementing
or Ca2+ accumulation activities at all. The 12 mutant
proteins, F368S, L370S, D371E, F372S, D375E, A377S, Y378A, V380S,
P381A, T382A, S383A, and G388A, had slightly lower complementing
activity than the wild-type protein, although these proteins seemed to
have essentially the same Ca2+ accumulation activity as the
wild-type protein. In contrast, S384A had lower Ca2+
accumulation activity, although it had essentially the same
complementing activity as the wild-type protein. The three severe
mutations, H4De, C373D, and C373R, had no dominant negative effects on
the viabilities of wild-type cells (data not shown), indicating that these mutations are loss-of- function ones.
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H3 and H4 May Not Be Required for the Localization of the Mid1 Protein-- The above results showing that the Mid1 proteins having a deletion of or a single mutation in H3 and H4 did not complement the mid phenotype suggest that these segments and amino acid residues are required for Mid1 function. However, it is also possible that those results are caused by mislocalization of the mutant proteins. To examine this possibility, the Mid1 protein fused with GFP at the C terminus, Mid1-GFP, was used for observing the protein localization by confocal fluorescence microscopy. The Mid1 protein is an integral membrane protein and present at the plasma membrane (1). It has also been shown that the Mid1 protein localizes at the endoplasmic reticulum (ER) membrane as well as the plasma membrane, as revealed by indirect fluorescence microscopy.2 The wild-type Mid1-GFP produced from low- and multi-copy plasmids has been shown to completely complement the mid phenotype and to localize at the plasma membrane and endoplasmic reticulum membrane normally,3 indicating that GFP and the overexpression has no effect on the activity and localization of the Mid1 protein. However, it should be noted that fluorescence images of Mid1-GFP produced from the low copy plasmid were very faint and hard to be photographed.3 In addition, we have tried to examine the localization of the Mid1 protein produced from its gene on the intrinsic chromosome or on a low copy plasmid by indirect fluorescence microscopy using polyclonal anti-Mid1 antibodies, but we failed because of the very small expression levels of Mid1. We therefore examined the localization of Mid1-GFP produced from the multicopy plasmid thereafter.
By using the gene encoding the wild-type Mid1-GFP on the multicopy
plasmid, the deletions or single mutations that had produced the mutant
phenotypes shown in Figs. 2 and 3 were introduced by exactly the same
methods as those used for the gene encoding the Mid1 proteins. Figs.
4A and 5A show that
the wild-type Mid1-GFP localized on the
plasma membrane and the endoplasmic reticulum membrane as expected,
confirming the previous results, and Figs. 4B and
5B show that GFP itself was present in the cytoplasm.
Fluorescence images for the localization of all the mutant Mid1-GFPs
tested were essentially the same as those of the wild-type Mid1-GFP
(Figs. 4 and 5), suggesting that the phenotypes of the mutant Mid1
proteins presented in Figs. 2 and 3 were not caused by the
mislocalization of the mutant Mid1 proteins.
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H3 and H4 Are Not Required for the Stability of the Mid1
Protein--
There is another possibility that the above results shown
in Figs. 2 and 3 were caused by a decrease in stability of the mutant Mid1 proteins. The contents of the mutant Mid1 proteins that have completely or partially lost the complementation activity were examined
by immunoblot analysis with antibodies to the GST-Mid1 protein. To
perform this analysis, we employed a very recently developed, improved
method of immunoblotting (28) which enabled us to examine the level of
the Mid1 proteins produced from the low copy plasmid used
in the complementation analysis shown in Figs. 2 and 3. The results
showed that the contents of the Mid1 proteins lacking the whole H3 or
H4 region and having single mutations in H3 or H4 were essentially the
same as those of the wild-type Mid1 protein (Fig.
6), indicating that the above possibility
is unlikely. We also confirmed that the mutant Mid1-GFPs described above were essentially the same level as the wild-type Mid1-GFP (data
not shown), suggesting that the fusion of GFP to Mid1 and the
expression of the fusants from the multicopy plasmid do not affect the
stability of the mutant proteins tested.
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DISCUSSION |
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We have shown in this study that the H3 and H4 segments are
necessary for Mid1 function. Deletion of either H3 or H4 resulted in a
complete loss of function. In addition, we individually evaluated the
amino acid residues in H3 and H4 required for the full activity of
Mid1. Before discussing the importance of these amino acid residues, we
will speculate about the structure of a putative Ca2+
channel composed of Mid1. Our previous work has shown that Mid1 might
form a homotetramer to make a channel (see supplemental material in
Ref. 2). In addition, Mid1 functions in an amiodarone-induced increase
in cytosolic Ca2+, whereas Cch1 does not (20). On the other
hand, it has been shown that the phenotypes of mid1 and
cch1 single mutants and a mid1 cch1 double mutant
are essentially the same (10, 17, 18), and immunoprecipitation
experiments have shown that Mid1 coprecipitates with Cch1 (10),
suggesting that Mid1 and Cch1 cooperate to work in some conditions. Two
possible structure-function relationships could be hypothesized. One is
that Mid1 might form a Ca2+ channel and serve a dual role
as a stretch-activated channel mediating Ca2+ influx and as
a stretch sensor, both of which might then generate a Ca2+
signal or a conformational change to activate a coupled
Ca2+ channel composed of Cch1. A mechanism analogous to
this is well elucidated in the coupling between dihydropyridine
receptors (the 1 subunits of voltage-gated Ca2+
channels) and ryanodine receptors (sarcoplasmic reticulum
Ca2+ release channels) in vertebrate skeletal muscles (36,
37). The other hypothesis is that Mid1 might form no channel by itself and instead be a regulatory subunit protein of a Ca2+
channel whose pore-forming subunit is Cch1. It is known that mammalian
voltage-gated Ca2+ channels have auxiliary subunits,
2
,
, and
, which regulate the activity of the pore-forming
1 subunit homologous to Cch1 (12-16). Because the structure of the
putative Ca2+ channel(s) composed of Mid1 and
Cch1 is an open question at present, we would like to explain the data
presented in this paper from the viewpoint of both hypotheses. In
particular, four severe loss-of-function mutations, D341E, F356S,
C373D, and C373R, and two mutations leading to a high level of
Ca2+ accumulation with a slightly low complementing
activity, G342A and Y355A, are discussed below.
Important Amino Acid Residues in H3
Asp341-- When Asp341 in H3 was replaced with Glu, Lys, Arg, Thr, Asn, Ala, or Leu, only the D341E mutation resulted in an almost complete loss of function, regarding maintenance of cell viability and Ca2+ accumulation, whereas the other mutations did not deteriorate the function of Mid1 (Fig. 2). We examined three independent strains containing the D341E mutation and obtained the same results. In addition, we confirmed that the D341E mutant gene did not contain an additional mutation elsewhere in the MID1 gene. Therefore, these results suggest that the size of the residues is not important, but an appropriate strength of negative charge of this position may be. Glu has a greater negative charge than Asp (pKa 4.25 versus 3.86), and thus Ca2+ would bind to Glu341 too tightly to pass the channel, if Mid1 is the pore-forming subunit. It has been reported that Asp substitution in the EEEE locus, known as the filter of the L-type Ca2+ channel, reduces ion selectivity by weakening ion binding affinity (38).
In terms of the second hypothesis that Mid1 is a regulatory protein of
a Ca2+ channel containing Cch1, Asp341 could be
an important amino acid residue that may interact directly or
indirectly with Cch1 and activate it. Coexpression study of the human
auxiliary subunit 2
with the
1 and
subunits has indicated
that
2
is required for an increase in the peak size of the N-type
Ca2+ current (13). Interestingly, although Mid1 has no
sequential similarity to the
2
subunit at all, both Mid1 and
2
are glycosylated plasma membrane proteins (1, 11, 39). Mid1 may
directly up-regulate Cch1 like
2
, and the loss-of-function
mutation D341E may lead Mid1 not to activate Cch1.
Gly342-- Cells of the G342A mutant have an interesting phenotype. They accumulate Ca2+ to a greater extent than wild-type cells and lose their viability (Fig. 2). One possibility is that the low viability is caused by much Ca2+ incorporated into this mutant. Because this mutation appeared not to affect the stability and localization of the G342A mutant protein (Figs. 4 and 6), the replacement of Gly342 with Ala may be sufficient to cause a conformational change, either activating Mid1 itself or up-regulating Cch1 through the mutated Mid1. It has been suggested that a Gly residue and its adjacent amino acid residues in the gate play important roles in the gating mechanism in several ion channels. For example, the most conserved Gly22 residue of E. coli MscL functions as the gate (40, 41). When Gly22 is replaced with hydrophilic residues, the resulting mutant MscL channels become more mechanosensitive. On the other hand, a Gly residue in a transmembrane segment can be also important for protein-protein interaction between two distinct membrane proteins. For example, Gly279, a central amino acid of the transmembrane segment 9 of Rh50 glycoprotein whose defect causes chronic hemolytic anemia and stomatocytosis, is important for the interaction with Rh30 protein in the plasma membrane of erythroid cell lineage (42, 43). By analogy, Gly342 in H3 may contribute to the interaction of Mid1 with Cch1. Although the two alternative hypotheses remain to be examined, the G342A mutation in Mid1 provides a unique experimental system to investigate the property of Mid1 regarding Ca2+ uptake.
Phe356-- The F356S mutation brought about a unique phenotype. The F356S protein does not complement the lethality of the mid1 mutant at all, but it still maintains the activity to accumulate Ca2+ close to a normal level (Fig. 2). It is, therefore, obvious that the death of the F356S mutant is not caused by a decrease in Ca2+ accumulation.
It has been shown that the C-terminal region downstream from the H4 segment is located in the cytoplasm (1). In addition, our recent study with protease protection experiments has shown that GFP in the Mid1-GFP fusion protein, in which GFP has been fused downstream from H3, is susceptible to protease added to intact yeast cells.3 In contrast, GFP in the Mid1-GFP fusion protein, in which GFP has been fused downstream from H4, is resistant to protease as expected.3 These results suggest that H4 is a transmembrane segment and that Phe356 is outside the plasma membrane. Therefore, we speculate that Phe356 interacts with an extracellular portion of other protein(s) which takes part in the maintenance of cell viability without regulating Ca2+ uptake. The F356S mutation would hardly affect the channel activity of Mid1 or the association of Mid1 with Cch1. In contrast, the Y355A mutation whose mutation site is neighboring to Phe356 resulted in a slight increase in Ca2+ accumulation. Thus, the region containing Tyr355 and Phe356 may be an active portion required for interaction with both Cch1 and another component necessary for viability.
H4 as a Possible Transmembrane Helix
In this study, 13 of 23 amino acid residues in H4 were found to be required for the maintenance of viability and 2 for Ca2+ accumulation, but the degree of decreases in viability or Ca2+ accumulation was small for all of the mutants but Cys373 (Fig. 3). It is therefore unlikely that H4 contributes to the formation of the filter or the gate whose amino acid substitution is supposed to cause a large change in Ca2+ permeability. Thus, a further study is to examine whether H4 is an inner or outer helix.
If Mid1 is a regulatory subunit protein of Cch1 according to the second hypothesis, most of amino acid residues in H4 would not be required for the interaction with and activation of Cch1. The C373D and C373R proteins completely lost Mid1 function regarding the viability and Ca2+ accumulation, but the C373A protein functioned quite normally. This indicates that Cys373 does not contribute to disulfide bonding (Fig. 3). Probably, the negative or positive charge of C373D and C373R interferes with the interaction between Mid1 and Cch1 or a protein regulating Cch1. It is also possible that the charges result in a serious conformational change leading to the inactivation of Mid1 not to activate Cch1.
In summary, using molecular genetic approaches, we have identified
amino acid residues and clusters in the H3 and H4 segments important
for Mid1 function. Especially, Asp341, Gly342,
Tyr355, Phe356, and Cys373 have
attracted a great deal of our attention. Further characterization of
mutations in these amino acid residues should result in a better understanding of the molecular basis of the function of Mid1 as well as
the structural and functional relationships between Mid1 and Cch1. In
addition, because it remains to be elucidated how the pore-forming
subunits of mammalian Ca2+ channels are regulated by
non-pore-forming auxiliary transmembrane subunits, those studies would
also shed light on the mechanism.
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ACKNOWLEDGEMENTS |
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We thank Masahiro Sokabe and Chikara Sato for advice on channel structure and function, Chikako Miyawaki for YEpMID1-GFP and YEpGFP, Akio Sugino for YEplac112, Philip Hieter for pRS315, Makiko Hoshi for a preliminary work, and Yumiko Higashi for secretarial assistance.
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FOOTNOTES |
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* This work was supported by CREST, Japan Science and Technology Corporation (to H. I.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The on-line version of this article (available at
http://www.jbc.org) contains Table II.
To whom correspondence should be addressed: Dept. of Biology,
Tokyo Gakugei University, 4-1-1 Nukui kita-machi, Koganei-shi, Tokyo
184-8501, Japan. Tel. and Fax: 81-42-329-7517; E-mail:
iida@u-gakugei.ac.jp.
Published, JBC Papers in Press, January 3, 2003, DOI 10.1074/jbc.M206993200
2 H. Yoshimura, S. Muto, and H. Iida, manuscript in preparation.
3 C. Miyawaki, H. Iida, H. Tatsumi, and M. Sokabe, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: GFP, green fluorescence protein; GST, glutathione S-transferase.
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REFERENCES |
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1. | Iida, H., Nakamura, H., Ono, T., Okumura, M. S., and Anraku, Y. (1994) Mol. Cell. Biol. 14, 8259-8271[Abstract] |
2. |
Kanzaki, M.,
Nagasawa, M.,
Kojima, I.,
Sato, C.,
Naruse, K.,
Sokabe, M.,
and Iida, H.
(1999)
Science
285,
882-886 |
3. |
Kanzaki, M.,
Nagasawa, M.,
Kojima, I.,
Sato, C.,
Naruse, K.,
Sokabe, M.,
and Iida, H.
(2000)
Science
288,
1347 |
4. | Carnero, E., Ribas, J. C., Garcia, B., Duran, A., and Sanchez, Y. (2000) Mol. Gen. Genet. 264, 173-183[CrossRef][Medline] [Order article via Infotrieve] |
5. | Baba, M., Baba, N., Ohsumi, Y., Kanaya, K., and Osumi, M. (1989) J. Cell Sci. 94, 207-216[Abstract] |
6. |
Ohsumi, Y.,
and Anraku, Y.
(1985)
J. Biol. Chem.
260,
10482-10486 |
7. |
Iida, H.,
Yagawa, Y.,
and Anraku, Y.
(1990)
J. Biol. Chem.
265,
13391-13399 |
8. | Fischer, M., Schnell, N., Chattaway, J., Davies, P., Dixon, G., and Sanders, D. (1997) FEBS Lett. 419, 259-262[CrossRef][Medline] [Order article via Infotrieve] |
9. | Paidhungat, M., and Garrett, S. (1997) Mol. Cell. Biol. 17, 6339-6347[Abstract] |
10. |
Locke, E. G.,
Bonilla, M.,
Liang, L.,
Takita, Y.,
and Cunningham, K. W.
(2000)
Mol. Cell. Biol.
20,
6686-6694 |
11. | Walker, D., and De Waard, M. (1998) Trends Neurosci. 21, 148-154[CrossRef][Medline] [Order article via Infotrieve] |
12. | Chien, A. J., and Hosey, M. M. (1998) J. Bioenerg. Biomembr. 30, 377-386[CrossRef][Medline] [Order article via Infotrieve] |
13. |
Gao, B.,
Sekido, Y.,
Maximov, A.,
Saad, M.,
Forgacs, E.,
Latif, F.,
Wei, M. H.,
Lerman, M.,
Lee, J. H.,
Perez-Reyes, E.,
Bezprozvanny, I.,
and Minna, J. D.
(2000)
J. Biol. Chem.
275,
12237-12242 |
14. |
Green, P. J.,
Warre, R.,
Hayes, P. D.,
McNaughton, N. C.,
Medhurst, A. D.,
Pangalos, M.,
Duckworth, D. M.,
and Randall, A. D.
(2001)
J. Physiol. (Lond.)
533,
467-478 |
15. |
Garcia, R.,
Carrillo, E.,
Rebolledo, S.,
Garcia, M. C.,
and Sanchez, J. A.
(2002)
J. Physiol. (Lond.)
545,
407-419 |
16. | Hanlon, M. R., and Wallace, B. A. (2002) Biochemistry 41, 2886-2894[CrossRef][Medline] [Order article via Infotrieve] |
17. |
Muller, E. M.,
Locke, E. G.,
and Cunningham, K. W.
(2001)
Genetics
159,
1527-1538 |
18. |
Bonilla, M.,
Nastase, K. K.,
and Cunningham, K. W.
(2002)
EMBO J.
21,
2343-2353 |
19. |
Matsumoto, T. K.,
Ellsmore, A. J.,
Cessna, S. G.,
Low, P. S.,
Pardo, J. M.,
Bressan, R. A.,
and Hasegawa, P. M.
(2002)
J. Biol. Chem.
277,
33075-33080 |
20. | Courchesne, W. E., and Ozturk, S. (2003) Mol. Microbiol. 47, 223-234[Medline] [Order article via Infotrieve] |
21. | Tokes-Fuzesi, M., Bedwell, D. M., Repa, I., Sipos, K., Sumegi, B., Rab, A., and Miseta, A. (2002) Mol. Microbiol. 44, 1299-1308[CrossRef][Medline] [Order article via Infotrieve] |
22. |
Maruoka, T.,
Nagasoe, Y.,
Inoue, S.,
Mori, Y.,
Goto, J.,
Ikeda, M.,
and Iida, H.
(2002)
J. Biol. Chem.
277,
11645-11652 |
23. | Sherman, F., Fink, G. R., and Hicks, J. B. (1986) Methods in Yeast Genetics , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
24. | Difco Laboratories. (1984) Difco Manual , 10th Ed. , pp. 1135-1141, Difco Laboratories, Detroit |
25. | Inoue, H., Nojima, H., and Okayama, H. (1990) Gene (Amst.) 96, 23-28[CrossRef][Medline] [Order article via Infotrieve] |
26. | Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd Ed. , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
27. | Ito, H., Fukuda, Y., Murata, K., and Kimura, A. (1983) J. Bacteriol. 153, 163-168[Medline] [Order article via Infotrieve] |
28. | Wu, M., Stockley, P. G., and Martin, W. J., Jr. (2002) Electrophoresis 23, 2373-2376[CrossRef][Medline] [Order article via Infotrieve] |
29. | Iida, H., and Yahara, I. (1985) Nature 315, 688-690 |
30. |
Cibulsky, S. M.,
and Sather, W. A.
(2000)
J. Gen. Physiol.
116,
349-362 |
31. |
Talavera, K.,
Staes, M.,
Janssens, A.,
Klugbauer, N.,
Droogmans, G.,
Hofmann, F.,
and Nilius, B.
(2001)
J. Biol. Chem.
276,
45628-45635 |
32. |
Nilius, B.,
Vennekens, R.,
Prenen, J.,
Hoenderop, J. G.,
Droogmans, G.,
and Bindels, R. J.
(2001)
J. Biol. Chem.
276,
1020-1025 |
33. | Jean, K., Bernatchez, G., Klein, H., Garneau, L., Sauve, R., and Parent, L. (2002) Am. J. Physiol. 282, C665-C672 |
34. |
Li, R. A.,
Ennis, I. L.,
French, R. J.,
Dudley, S. C., Jr.,
Tomaselli, G. F.,
and Marban, E.
(2001)
J. Biol. Chem.
276,
11072-11077 |
35. |
Penzotti, J. L.,
Lipkind, G.,
Fozzard, H. A.,
and Dudley, S. C., Jr.
(2001)
Biophys. J.
80,
698-706 |
36. | Catterall, W. A. (1991) Cell 64, 871-874[Medline] [Order article via Infotrieve] |
37. | Rios, E., Pizarro, G., and Stefani, E. (1992) Annu. Rev. Physiol. 54, 109-133[CrossRef][Medline] [Order article via Infotrieve] |
38. |
Cloues, R. K.,
Cibulsky, S. M.,
and Sather, W. A.
(2000)
J. Gen. Physiol.
116,
569-586 |
39. |
Gee, N. S.,
Brown, J. P.,
Dissanayake, V. U.,
Offord, J.,
Thurlow, R.,
and Woodruff, G. N.
(1996)
J. Biol. Chem.
271,
5768-5776 |
40. | Batiza, A. F., Rayment, I., and Kung, C. (1999) Struct. Fold Des. 7, R99-R103[Medline] [Order article via Infotrieve] |
41. |
Ou, X.,
Blount, P.,
Hoffman, R. J.,
and Kung, C.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
11471-11475 |
42. |
Huang, C. H.,
Liu, Z.,
Cheng, G.,
and Chen, Y.
(1998)
Blood
92,
1776-1784 |
43. |
Hyland, C. A.,
Cherif-Zahar, B.,
Cowley, N.,
Raynal, V.,
Parkes, J.,
Saul, A.,
and Cartron, J. P.
(1998)
Blood
91,
1458-1463 |
44. | Gietz, R. D., and Sugino, A. (1988) Gene (Amst.) 74, 527-534[CrossRef][Medline] [Order article via Infotrieve] |
45. |
Sikorski, R. S.,
and Hieter, P.
(1989)
Genetics
122,
19-27 |