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J Biol Chem, Vol. 273, Issue 44, 28912-28920, October 30, 1998
From the Laboratory of Biochemistry and Genetics, NIDDK, National
Institutes of Health, Bethesda, Maryland 20892-0830
Mak21-1 mutants are unable to
propagate M1 double-stranded RNA, a satellite of the L-A
double-stranded RNA virus, encoding a secreted protein toxin lethal to
yeast strains that do not carry M1. We cloned
MAK21 using its map location and found that Mak21p is
homologous to a human and mouse CAATT-binding protein and open reading
frames in Schizosaccharomyces pombe and
Caenorhabditis elegans. Although the human protein
regulates Hsp70 production, Mak21p is essential for growth and
necessary for 60 S ribosomal subunit biogenesis. mak21-1
mutants have decreased levels of L-A coat protein and L-A
double-stranded RNA. Electroporation with reporter mRNAs shows that
mak21-1 cells cannot optimally express mRNAs which,
like L-A viral mRNA, lack 3'-poly(A) or 5'-cap structures but can
normally express mRNA with both cap and poly(A). The virus propagation phenotype of mak21-1 is suppressed by
ski2 or ski6 mutations, each of which
derepresses translation of non-poly(A) mRNA.
The L-A dsRNA1 virus has
a single segment of 4.6 kilobases which replicates inside yeast cells,
where it is maintained at a copy number of 1,000 or more without a
substantial adverse effect on cell growth (1, 2). The icosahedral L-A
virus particles are composed of 120 copies of the major coat protein
(Gag) and about 2 copies of a Gag-Pol fusion protein (3, 4). The L-A (+) strand serves as the mRNA, encoding Gag and Gag-Pol, the latter formed by a Because L-A mRNA lacks both a 5'-cap and 3'-poly(A) and induces a
Mutations in genes resulting in an inability to propagate
M1 dsRNA are called mak mutations. 20 mak genes have been found to affect or be deficient in 60 S
ribosomal subunits (21-23), but no mak mutants deficient in
40 S subunits were identified (22). Based on the proposal that the
3'-poly(A) structure may facilitate the 60 S subunit joining reaction
(24), we suggested that 60 S-deficient mutants may be preferentially
defective in translation of non-poly(A) mRNAs, such as viral
mRNA (10, 22). However, there has been no direct evidence for this
suggestion.
Here we clone the MAK21 gene and analyze the effects of
mak21 mutations on translation and ribosome subunits.
Cloning of MAK21
Saccharomyces cerevisiae strain D2-3C (a
mak21-1 ura3 his5 ade2) was provided by Daniel Masison.
Strains 3164 (a arg1 kar1-1 L-A-HN M1) and
3165 ( Subcloning of MAK21
Subclones of The XbaI-NheI fragment from p782 encompassing
MAK21 was ligated into the XbaI site of pRS315
forming pH102 (=pRS315-MAK21). MAK21 bordered by
XhoI and SacI sites was then transferred to the
XhoI-SacI window of pRS313 and pRS316 (28),
resulting in pH110 and pH107, respectively.
mak21::HIS3 Disruption
The XbaI-SalI fragment from p782 including
MAK21 was ligated into
XbaI-SalI-digested pBS KS+. After ligation of a
BglII linker (NEB1001) into the StuI site at base
3447 of MAK21, the HIS3 gene of pJJ215 (29),
bordered by BamHI sites, was cloned into the BglII-digested MAK21 plasmid, resulting in pH75
(Fig. 1 shows the region deleted). To confirm the disruption, genomic
DNA of putative disruptant strain YHE353 was digested with
SnaBI and PstI and probed with the
SalI-PstI fragment from p782. This confirmed the
integrity of the 3'-integration border. Probing YHE353 genomic DNA
digested with SnaBI and NsiI with the
BamHI-NsiI fragment from pJJ215 confirmed the
integrity of the 5'-integration site.
pRS316-SKI2 (30) and pRS316-SKI6 (11) have been described.
ADH1-luciferase and SSA3-luciferase Plasmids
The expression plasmids pH116 and pH118 were made by ligating
the ADH1 cassette from pVT103 (31), obtained after PCR
amplification using primers HE66 (5'-ACAGCTAGCATTACGCCAGCAACTTCT-3')
and HE67 (5'-ACAAGATCTTAATGCAGCCGGTAGAG-3'), into
PvuII-digested pRS313 (28), replacing the latter's multiple
cloning region. In pH118 the ADH1 cassette is oriented
opposite that in pH116, being directed toward the HIS3 gene.
The luc+ ORF, bordered by BamHI and
XhoI sites, was transferred from pH96 (see below) into the
BamHI-XhoI windows of pH116 and pH118, creating
pH170 and pH171. Thus pH170 and pH171 have the
ADH1-luciferase cassette in two orientations relative to
HIS3. A 516-base pair SSA3 promoter fragment was
obtained by PCR amplification from the genomic DNA of strain L-A-luciferase Hybrid Plasmids, pH149 and pH161, and Other
Constructs
To place the L-A genome under control of the Sp6 promoter, the
L-A gag region was amplified by PCR from pTIL05 (5) using oligonucleotides JR71
(5'-CGACGGCCAGTGAATTCATTTAGGTGACACTATAGAAAAATTTTTAAATTC-3') and
HE4 (5'-CTTGGATCCCGGGTCCGTGTGAGGGTTC-3'). The product was cloned into
the HincII site of pBS KS+ (pH91). The
EcoRV-BamHI fragment from pH91 was replaced with
the EcoRV-BamHI fragment from pTIL05, producing
pH94.
An SmaI site was introduced at the L-A ATG codon of pH94 by
site-directed mutagenesis using oligonucleotide HE54
(5'-ACAAATCTTAGCCCGGGGAGTTATAT-3'), resulting in pH148. The
SphI site of pH94 was changed into an SmaI site
by linker insertion using oligonucleotide HE52 (5'-ACCCGGGTCATG-3'), resulting in pH160.
To place luc+ between StuI sites, the
luc+ ORF was amplified by PCR from plasmid
pSP-luc(+) (Promega) using oligonucleotides HE39
(5'-CAAAGGCCTCATGGAAGACGCCAAAAAC-3') and HE40
(5'-CACAGGCCTAAGCTTCACTTACACGGCGATCTTTCC-3'). The product was cloned
into the EcoRV site of pBS KS+, resulting in clone pH96.
Insertion of the StuI fragment from pH96 containing luc+ into the SmaI site of pH148 and pH160
resulted in pH149 and pH161, respectively.
A human CCAAT box-binding factor (hCBF) clone (pMT2-CBF) was kindly
provided by Daniel Linzer (Department of Biochemistry, Molecular
Biology, and Cell Biology, Northwestern University). The hCBF ORF is
tightly bordered by NcoI sites. The NcoI fragment of pMT2-CBF was cloned into pH291, producing pH293, in which hCBF expression is under the control of the ADH1 promoter present
on this LEU2 2-µ plasmid.
GAL-promoted MAK21 Plasmid Construction
An XhoI site was introduced by site-directed
mutagenesis (Bio-Rad) immediately downsteam of the MAK21
stop codon in pH115 (see below) using oligonucleotide HE81
(5'-GTGTTTAGTTAATGTATTATCTCGAGCTAGTCTGAATCTTG-3'), resulting in clone
pH183. The HindIII-XhoI-bordered MAK21
ORF was transferred into the HindIII-XhoI window
of pBC KS+ (Stratagene) to give pH232. The ADH1 expression
cassette from pVT101 was amplified by PCR using oligonucleotides HE66
and HE67 and ligated into PvuII-digested pRS315, resulting
in pH268.
The GAL1,10 promoter bordered by EcoRI and
BamHI sites was transferred from pBM272 (32) into the
EcoRI-BamHI window of pBC KS+ (Stratagene). After
the EcoRI site was changed into an NheI site
using of oligonucleotide HE93, and the BamHI site was
changed into a HindIII site, the resulting
NheI-HindIII fragment encompassing the
GAL1 promoter was used to replace the
NheI-HindIII-bordered ADH1 promoter in
pH268. The resulting construct was designated pH272, and the
MAK21 ORF encompassed within the
HindIII-XhoI fragment of pH232 was ligated into
its HindIII-XhoI window, resulting in pH287.
MAK21 Deletion Constructs
The SmaI-ApaI sites of pH102
(=pRS315-MAK21) were fused, resulting in pH113. Using oligonucleotide
HE55 (5'-TTCTCACTCATTAAGCTTGATTACTGTAC-3') an HindIII site
was created in pH113 immediately upstream of the ATG start codon of
MAK21, resulting in pH115.
MAK21 5'-Deletions--
Ser-207, Val-228, or Arg-248 was changed
into an ATG start codon with a HindIII site directly
upstream of Ser-207, Val-228, or Arg-248 in pH115 by site-directed
mutagenesis using oligonucleotide HE107
(5'-GGAATAATAAGTTTGTCAGACATAAGCTTAGTAGTTTGAGTAAT-3') or HE106
(5'-CATCATCATTTTGTCCCATAAGCTTATCCAATGGAATTTC-3') or HE85 (5'-TAGCGTTTGCTTACCCATAAGCTTAAGTTTCTCAATCTG-3'). During these mutageneses an XhoI site was created directly downstream of
the MAK21 stop codon using oligonucleotide HE81
(5'-GTGTTTAGTTAATGTATTATCTCGAGCTAGTCTGAATCTTG-3'). Subsequently the
HindIII sites were fused, producing pH310, pH311, and pH216,
respectively.
MAK21 3'-Deletions--
Asn-961, Ala-933, Val-898, or Ala-873
was changed to a stop codon with an XhoI site directly
downstream of Asn-961, Ala-933, Val-898, or Ala-873 in clone pH115 by
site-directed mutagenesis using oligonucleotide HE75
(5'-CGAAGGAACGTTTTTTCTCGAGTTAGTCTTGTTCTCCATCGAA-3'), HE88
(5'-TTCTTGGCTGCCTTCCTCGAGTTAATCTTCATCATCAAT-3'), HE89
(5'-ACTGTCGTCGTCATCCTCGAGTTAATCTGGTCTCGATTT-3'), or HE113
(5'-TTCGTCATCACTATCGAACTCGAGTTATTTGTTAGATTTCTTTCCT-3'). During these mutageneses a second XhoI site was
created downstream of the MAK21 stop codon using
oligonucleotide HE81. Subsequently the XhoI sites were
fused, resulting in pH211, pH280, pH281, and pH309.
Strain Constructions
To verify that M1 dsRNA is lost from strains from
which polysome profiles would be prepared a mak21-1 strain
(YHE414 = MAT On crossing YHE561 (MATa leu2 trp1 his3 ski6-2 K+) with
YHE414, the slow growth phenotype of mak21-1 (26) segregated
2:2 as did the inability to grow at 39 °C resulting from the
ski6-2 allele (11). Among 28 tetrads dissected, three showed
4 K+:0 segregation, 21 showed 3 K+:K Similarly, in the cross of YHE626 (MATa leu2 trp1
ski2::HIS3 K+) with YHE414 segregants with
mak21-1 were identified by their slow growth phenotype and
ski2 by His prototrophy. Of 24 tetrads, four showed 4 K+:0
segregation, 11 showed 3 K+:1 K Strains YHE561, YHE414, YHE618, YHE626, YHE633, and YHE686 were grown
in -Leu, -Ura medium until the cell density reached an
A600 between 0.400 and 0.550. Cells were broken
with glass beads in phosphate-buffered saline containing 100 µg/ml
phenylmethylsulfonyl fluoride. The protein concentration was measured
with the Bradford assay (Bio-Rad), and 7.5 µg of total protein was
analyzed for Gag by Western blotting. From the extract dsRNA was
isolated by phenol extraction.
Western Blotting
Protein extracts were analyzed by electrophoresis on an 8%
SDS-polyacrylamide gel and blotted onto a nitrocellulose membrane. L-A
Gag protein was visualized using an anti-Gag monoclonal antibody (yCP6)
and anti-mouse IgG-conjugated alkaline phosphatase (Promega) (33).
Gfp-Mak21p Fusion Protein Constructs and Localization
A 5'-MAK21 sequence tag was fused to the 3'-end of
the green fluorescent protein (GFP) ORF by amplifying the GFP ORF from plasmid pYGFP (34) using primers HE57
(5'-GCCGTTGTTCTCACTCATTTTGTACAATTCATCCATACC-3') and HE58
(5'-AACAAGCTTAATGTCTAAAGGTGAAGAAT-3'). This product was used as a
megaprimer to amplify the first 565 nucleotides from MAK21
from plasmid p782 aided by oligonucleotides HE58 and HE59 (5'-TCTCTGCAGATTCTGATT-3'). The product was cloned into the
EcoRV site of pBC KS+ (Stratagene) creating pH165. The
HindIII/PstI fragment from pH165 was used to
replace the HindIII/PstI window of pH115,
creating pH209. Strain YHE393 carrying pH209 is strain YHE505.
M827 was changed to a stop codon as described for pH211 but using
oligonucleotides HE83 (5'-GTTCACGGGACCCTGCTCGAGTTAAATATCAGATGCCTTAA-3') and HE81, resulting in pH212. The HindIII-PstI
fragment from pH165 was used to replace the
HindIII-PstI window of pH212, creating pH283.
YHE393 carrying pH283 is strain YHE533.
The GFP ORF from pYGFP was amplified by PCR using oligonucleotides HE58
and HE69 (5'-AGTCTCGAGTTATTTGTACAATTCATCCATAC-3'). The product was
cloned into the EcoRV site of pBC KS+, resulting in pH163.
Replacement of the HindIII-XhoI bordered MAK21
ORF from pH183 with the HindIII-XhoI-bordered GFP
ORF from pH163 resulted in pH210. YHE393 carrying pH210 is strain
YHE506.
GFP was visualized using an Axiovet 100TV microscope (Zeiss, Germany),
and images were recorded using a C5810 color chilled 3CCD camera
(Hamamatsu Photonics Systems, Japan) and processed using Adobe
Photoshop.
Cloning of MAK21--
The mak21-1 mutation is on
chromosome IV between trp1 and aro1 (26).
Because the rrp1 mutation maps close to mak21-1
and, like mak21-1, rrp1 results in loss of M1
dsRNA, it was thought possible that RRP1 was identical to
MAK21 (35). However, the restriction map of the clones
carrying RRP1 are different from that of MAK21, and recently RRP1 has been sequenced, showing that it is
identical to
YDR087C3 and thus
distinct from MAK21.
MAK21 Is Essential--
One of the MAK21 genes of the
diploid strain YHE305 (MATa/MAT
To investigate if MAK21 is needed for spore germination or for cell
growth, tetrads from strain YHE353 carrying pH107 (URA3 CEN
MAK21, see "Materials and Methods") were were germinated on medium lacking uracil. Cells from eight tetrads containing four germinating ascospores were transferred to media containing
5-fluoroorotic acid to eliminate the URA3 plasmid. All eight
tetrads showed 2:2 segregation for growth. Thus MAK21 is not
only needed for spore germination, it is absolutely required for
viability.
Mak21p Homology with hCBF--
The 1,025-residue Mak21p shares
substantial sequence similarity throughout its length with the hCBF
(36). Other ORFs of unknown function from mouse, Caenorhabditis
elegans, and Schizosaccharomyces pombe also show
significant amino acid similarity with Mak21p (see Fig.
2). The hCBF stimulates expression from
the hsp70 CCAAT box promoter in tissue culture cells. It was identified
by its specific binding to this promoter. As shown diagramatically in Fig. 2, 77 residues are conserved in all five homologs, and 90 are
conserved in four of five homologs, and these are distributed through
most of the length of the five proteins.
Mak21p Is Not Required for Yeast Hsp70 (SSA3) Expression--
The
control of human hsp70 expression by hCBP suggested that the homologous
Mak21p might control the expression of yeast hsp70. The most homologous
yeast hsp70 to the human hsp70 controlled by hCBP is Ssa3p (37, 38). Of
its 649 residues, 445 are identical to the human hsp70. The promoter of
the SSA3 gene contains an inverted CCAAT box.
To estimate expression from the SSA3 promoter, we
constructed two plasmids (pH172 and pH173) in which transcription of
luciferase was driven by the SSA3 promoter, differing only
in the orientation of the SSA3-luciferase cassette relative
to the HIS3 gene. Two similar plasmids, in which the
ADH1 promoter drives luciferase transcription, were used as
controls (see "Materials and Methods"). The four plasmids were
transformed into strain YHE414 (MAT
For luciferase expression from the ADH1 promoter, the ratio
of activity in a mak21-1 mutant to that in the wild type was
1.57 ± 0.46 (10 independent assays), probably not a significant
increase. For luciferase from the SSA3 promoter, the same
ratio was 4.7 ± 3.4. The increase in SSA3-promoted
activity in mak21 cells is the opposite of what would be
expected if Mak21p were functioning the same in yeast as does hCBF in
human cells.
hCBF Does Not Complement a mak21 Deletion--
From transformants
of a clone of hCBF under the ADH1 promoter
(pH293-LEU2 2 µ) in YHE393 (MAT L-A Levels Are Reduced in mak21-1 Strains--
The same eight
strains used to examine SSA3 expression above were used to
compare L-A levels in mak21-1 and MAK21 strains. L-A Gag levels are at least 2-fold less in the mak21-1
strains compared with the MAK21-complemented strains (Fig.
3). Examination of phenol-extracted
nucleic acids or RNase-resistant dsRNA showed likewise that there was
twice as much L-A dsRNA present in the extract of MAK21
cells as in that of mak21-1 cells (Fig. 3).
Mak21p Is Involved in 60 S Ribosomal Subunit
Biogenesis--
Polysome profiles (10, 11) were examined from
wild-type strain YHE413, the mak21-1 strain YHE414, YHE432
(YHE414 containing plasmid pRS313 {HIS3 CEN}) and YHE433
(YHE414 containing plasmid pH110 {HIS3 CEN MAK21}).
Strains YHE413 and YHE414 were grown in YPAD, and strains YHE432 and
YHE433 were grown in -His medium. The polysome profiles of the
mak21-1 strains YHE414 and YHE432 show a marked increase in
the ratio of free 40 S to 60 S ribosomal subunits compared with the
wild-type strain YHE413 and also contain pronounced halfmer polysomes
peaks (Fig. 4A). The
40 S:60 S ratio was restored to wild-type levels by the
MAK21 expression plasmid (strain YHE433), with a concurrent
disappearance of halfmer polysomes.
Because the ratio of free 60 S subunits to free 40 S subunits was
decreased in mak21-1 strains, we examined the ratio of total 40 S to 60 S subunits. The mak21-1 strain YHE432
containing the vector pRS313 was compared with the same strain carrying
the MAK21 plasmid pH110 grown in -His medium to an
A600 between 0.4 and 0.5. Cells were broken in
buffer lacking cyclohexamide and MgCl2, and the cell
extract was loaded onto a sucrose gradient lacking MgCl2.
The sucrose gradients were centrifuged for 14 h at 27,000 rpm. In
the mak21-1 strain the 40 S:60 S ratio (as
A260) was 0.83, whereas this ratio was 0.64 in
the isogenic MAK21 strain (Fig. 4B). Thus the
mak21-1 strain has an imbalance in the 40 S:60 S ratio
probably because of a decrease in 60 S ribosomal subunits since the
40 S peaks are nearly identical in the two strains whereas the 60 S
peaks differ.
Mak21p Depletion and Overexpression--
Because MAK21
is essential for cell viability we analyzed the effect of depleting
Mak21p on ribosomal subunit biogenesis by putting Mak21p expression
under the control of the GAL1 promoter. After transforming
pH287 (GAL1::MAK21) into strain YHE393
(MAT
Strain YHE565 was grown in galactose/raffinose medium to
A600 = 0.640. Cells were collected and
resuspended in YPAD, to shut off production of Mak21p, or in
YPAgal/raf. The doubling time in galactose/raffinose medium was 3 h and 20 min, but the initial doubling time after transfer to glucose
medium was decreased to 2 h and 15 min. After 10 h of growth
in dextrose the doubling time started to increase and was 10 h and
45 min after approximately 40 h of growth.
Polysomes were prepared from strain YHE565 grown for 20 h in
glucose medium with a final A600 of 0.450 and
final doubling time of 7.5 h. The polysome profile showed an
extremely large 40 S ribosomal subunit peak (Fig.
5).
Potential MAK21 overexpression constructs included high copy
(2 µ) plasmids in which expression of the intact MAK21
ORF, the NH2 terminus (Lys-507 = stop), or the COOH
terminus (Asn-509 = start) is driven by the ADH1
promoter. Strain YHE413 transformed with these constructs in each case
maintained the satellite virus M1 and had normal polysome
profiles.
mak21-1 Selectively Reduces Expression of mRNAs Lacking 5'-Cap
or 3'-Poly(A)--
Luciferase expression of mRNAs carrying a
5'-cap, a 3'-poly(A) tail, or both or neither was examined by
electroporation into isogenic mak21-1 and wild-type strains
(Table I). The absence of either 5'-cap
or 3'-poly(A) resulted in a modest, but consistent, decrease in
expression in mak21-1 cells. To assess whether viral mRNA translation was affected, we constructed plasmids in which luciferase was imbedded in-frame at various points in the L-A sequence,
and mRNAs lacking both cap and poly(A) (like viral mRNA) were
electroporated into mak21-1 and MAK21 cells. The
SP6 promoter was used to produce these mRNAs with an authentic
5'-end.
As shown in Table II, for luciferase
inserted at the initiator AUG of the L-A sequence and at base 1834, the
mak21-1 mutation results in a consistent decrease of about
2-fold in expression of these mRNAs. In other experiments, the same
results were obtained with luciferase inserted at nucleotide 197, 464, or 971 of the L-A sequence (data not shown). Thus there is a clear
decrease in translation in mak21-1 cells, compared with
MAK21 cells, when the mRNAs are less competitive,
i.e. if they lack a cap and a poly(A) tail.
MAK21 5'- and 3'-Deletion Mutants and Their
Polysomes--
Although the NH2-terminal region of Mak21p
is not homologous to that of hCBF and is absent from the S. pombe homolog, the NH2-terminal region of hCBF has the
ability to interact with p53 and the adenovirus E1A transcriptional
activator (39). There is likewise little amino acid similarity among
the COOH-terminal regions of Mak21/hCBF-like proteins, even between the
human and mouse proteins. However, all of the Mak21/hCBF-like proteins
have an abundance of lysine residues in this region. It has been
postulated that hCBF contains a nuclear localization signal in this
part of the protein. Several 5' and 3' MAK21 deletions were constructed and transformed into strain YHE393 (MAT
The first 207 amino acids of Mak21p are completely dispensable for its
function (Fig. 5B). Up to 228 amino acids can be deleted from the NH2 terminus before the Mak21p function is
partially debilitated (Fig. 5C). Thus in contrast to hCBF
there does not seem to be an important role for the Mak21p
NH2 terminus. This is in agreement with the fact that this
whole region is absent in the S. pombe homolog (Figs. 1 and
2). Deleting 64 amino acids from the COOH terminus already causes some
loss of function (Fig. 5E). However, 127 amino acids can be
deleted from the COOH terminus before Mak21p is seriously debilitated
(Fig. 5F). Thus a substantial part of the lysine-rich region
postulated to be involved in nuclear localization can be deleted.
Localization of Gfp-Mak21p and Gfp-Mak21p
3'-Deletions--
Because hCBFp and Mak21p are involved in nuclear
processes, transcriptional activation of HSP70 genes and 60 S
ribosomal subunit biosynthesis, respectively, we investigated the
cellular localization of Mak21p. GFP was fused to the NH2
terminus of Mak21p (pH209). This fusion protein was capable of
complementing a mak21 ski6-2 and ski2
The effect of a mak21-1 mutation combined with either a
ski6-2 or a ski2 The mak21-1 mutation was isolated because it lost the
M1 dsRNA satellite of the L-A virus. We show here that
MAK21 is identical to YDR060W encoding a homolog of the
hCBF. The hCBF was isolated based on its ability to bind specifically
to the CCAAT site of the hsp70 promoter, and hCBF apparently regulates
the expression of hsp70 in tissue culture cells. S. cerevisiae has a very close homolog of human hsp70, Ssa3p. In a
mak21-1 strain, we find that expression from the
SSA3 promoter is not decreased as expected but is actually
increased. The observed result may be due to some "stress"
resulting from the slow growth of mak21-1 cells.
SSA3 transcription is increased in early stationary phase
(38), and the slow growth of mak21-1 cells may mimic this
condition. In addition, the NH2-terminal part of hCBF which
interacts with p53 and E1A is not essential for the yeast gene. A
substantial part of the COOH-terminal lysine-rich region believed to be
involved in nuclear localization is likewise dispensable.
We find that mak21-1 results in a decrease in the level of
free 60 S ribosomal subunits and that cells starved of Mak21p have very low levels of free 60 S particles and a large amount of free 40 S subunits. Because MAK21 is an essential gene, it is
likely that its essential role is related to its role in ribosome
biogenesis. Perhaps Mak21p is necessary for the transcription of some
component necessary for 60 S ribosome biogenesis, but such a putative
target gene remains to be identified. A fusion of GFP with Mak21p was found to localize in a single small central region of unfixed cells but
was more diffuse in fixed cells, precluding confirmation of our
suspicion that this was the nucleolus. Similar behavior has been
reported for Nip7p, another protein involved in 60 S ribosome
biogenesis (41).
We have found previously that mutations in many genes resulting in
deficiency in free 60 S ribosomal subunits result in loss of
M1 dsRNA (21-23). Here we have used electroporation of
mRNAs to investigate the connection between deficiency of 60 S
subunits and loss of M1 dsRNA. We find that mRNAs
lacking 5'-cap or 3'-poly(A) or both are translated about 2-fold less
well in mak21-1 strains than in isogenic wild-type cells.
Because the L-A mRNA lacks both 5'-cap and 3'-poly(A) (42, 43), it
is expected that translation of L-A mRNA should be impaired in
mak21-1 cells. In agreement with this result, the copy
number of L-A virus, as judged by the levels of Gag protein or of L-A
dsRNA, is about half the normal level in mak21-1
strains.
Why does a 2-fold decrease in L-A expression result in complete loss of
M1? We have suggested previously that L-A has limited cis-packaging, meaning that the L-A genome is preferentially
encapsidated in the coat proteins that it encodes (17, 44). This
implies that satellite dsRNAs, such as M1, can only use the
excess coat proteins produced from the L-A mRNA for their
propagation. One particularly striking example is X dsRNA, a deletion
mutant of L-A which lacks most of the Gag and Pol coding
sequences but retains the sites on the RNA necessary for packaging,
replication, and transcription of L-A (44-46). Although the parent
molecule, L-A, is not lost in mak mutants that are 60 S
subunit-deficient, X dsRNA is lost in these strains (44). A decrease in
the efficiency with which L-A is translated would preferentially affect
X or M1. Here we show that in a mak21-1 strain,
translation of any of several L-A-like mRNAs is indeed impaired if,
as for the L-A mRNA itself, the 5'-cap and/or 3'-poly(A) is
missing, but not if, as for cellular mRNAs, both cap and poly(A)
are present.
These results explain why mak mutants deficient in
translation of L-A lose M1 dsRNA, but do not lose L-A
dsRNA. It is likely that the deficiency of free 60 S subunits seen in
mak21-1 strains is the cause of its inefficient translation
of mRNAs lacking 5'-cap or 3'-poly(A) or both. However, the exact
mechanism by which 60 S deficiency leads to this poor translation
remains controversial. It has been suggested that the 3'-poly(A)
structure is involved in joining of 60 S subunits to the 40 S complex
waiting at the initiator AUG (47). In this model, a deficiency of 60 S
subunits would be expected to favor poly(A)+ mRNAs over
poly(A) We thank Daniel Linzer, Northwestern
University, for a clone of hCBF. We thank John Hanover and Dona Love
for help with fluorescence microscopy.
*
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.
§
To whom correspondence should be addressed: Bldg. 8, Rm. 225, NIH,
8 Center Dr. MSC0830, Bethesda, MD 20892-0830. Tel.: 301-496-3452; Fax:
301-402-0240; E-mail: wickner{at}helix.nih.gov.
The abbreviations used are:
dsRNA, double-stranded RNA; PCR, polymerase chain reaction; ORF, open reading
frame; hCBF, human CCAAT-binding factor; GFP, green fluorescent
protein.
2
L. Benard, D. C. Masison, and R. B. Wickner,
submitted for publication.
3
D. R. Stanford, S. M. Hess, R. J. Crouch, and A. K. Hopper, personal communication.
Mak21p of Saccharomyces cerevisiae, a Homolog of
Human CAATT-binding Protein, Is Essential for 60 S Ribosomal
Subunit Biogenesis*
, and
ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
1 ribosomal frameshift event (5, 6). These proteins also
support the propagation of a satellite RNA, called M1
dsRNA, which encodes the secreted polypeptide "killer toxin" and
immunity to this toxin (7). Propagation of M1 is
particularly sensitive to the efficiency of expression of the L-A
mRNA, suggesting that only excess Gag and Gag-Pol, above the needs
of L-A itself, are available to support M1.
1 ribosomal frameshift in expressing Gag-Pol, mutations affecting L-A
virus propagation have included mutations in many components of the
translation apparatus. Mutations in SKI2, SKI3, SKI6, SKI7, and SKI8 result in
elevated virus copy number (8, 9) because of enhanced translation of
mRNAs lacking a 3'-poly(A) structure (10,
11).2 Mutation of
SKI1 enhances viral expression (8) because of a defect in a
5' 
3' exoribonuclease that degrades uncapped RNAs (such as viral
mRNA) (12-15). The efficiency of L-A's programmed ribosomal
frameshifting is affected by mutations in 5 S rRNA (16) as well as by
several other genes and drugs influencing translation (17-20). Most of
these agents and mutations accordingly influence viral propagation.
MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
arg1 thr(1,x) kar1-1 L-A-HN M1)
were used for cytoduction of L-A and M1. Yeast genomic 
clones (25), ATCC71164, 70347, 70812, 70443, 71181, and 71075 were
obtained from the American Type Culture Collection. Pfu
polymerase (Stratagene) was used for PCR and a Bio-Rad kit for
site-directed mutagenesis.
clones covering the region on chromosome IV where
mak21-1 is located (26) were tested for complementation of
mak21-1 using in vivo recombination with
vector pBM2240 (27). Strain D2-3C was transformed by the spheroplast
method with a mixture of 1 µg of clone DNA and 0.1 µg of linear
plasmid pBM2240 digested with EcoRI and XhoI.
These transformants were used as cytoduction recipients from strain
3165, and the cytoductants were tested by the killer assay (9). Only
clone 71164 complemented mak21-1. Media have been defined
previously (9).
ATCC71164 in the centromere plasmid pRS316 were
tested for complementation of mak21-1 in strain D2-3C
cytoduction as above. Only the 6.8-kilobase XbaI fragment
shown in Fig. 1 complemented mak21-1. Subclones of this
fragment inserted in pRS316 (Fig. 1) included the 4.7-kilobase
XbaI-SalI fragment (p782) which complemented
mak21-1.
1278b
using primers HE49 (5'-CTTGGATCCTTTGTAGCGTTTAGTACC-3') and HE50
(5'-CAAGCTAGCATTGCCTGAACAATACTC-3'). This SSA3 fragment was
used to replace the ADH1 promoter in pH170 and pH171,
resulting in pH172 and 173, respectively.
ura3 his3 mak21-1) was crossed with a
wild-type strain (YHE413 = MATa leu2 his3 K+), and the
ability to maintain M1 satellite RNA was shown to segregate
2:2 in all eight meiotic tetrads was examined.
, three showed 2 K+:2 K, and
one showed 1 K : 3 K+. One segregant, strain YHE618 (69-15D = MATa ura3 leu2 his3 mak21-1 ski6-2 K+ ts39 °C), was
transformed with pRS316-SKI6 (11) and became K (YHE659). This K
strain was then allowed to lose pRS316-SKI6 and transformed with
plasmids carrying SKI6 and/or MAK21.
, and 9 showed 2 K+:2 K. One
segregant, YHE633 (71-4D = MATa ura3 leu2 trp1 mak21-1
ski2::HIS3 K+), was transformed with pRS316-SKI2 and
became K as a result (YHE658). This strain was allowed to lose
pRS316-SKI2 and transformed with plasmids carrying SKI2
and/or MAK21.
RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
clones of this region (25) were screened for complementation of
mak21-1, and only ATCC71164 showed positive complementation (see "Materials and Methods"). Subclones of 71164 were likewise tested for complementation of mak21-1, and only the
6.8-kilobase XbaI fragment shown at the top of
Fig. 1 complemented mak21-1. Subcloning this XbaI fragment showed that the
KpnI, EagI, and SpeI sites were within
the complementing gene (Fig. 1). Sequencing the ends of the
XbaI-SalI fragment showed that the
XbaI site on the left in Fig. 1 is at base
570,079 of chromosome IV, and the XbaI site at the
right is at base 576,879. The KpnI,
EagI, and SpeI sites are all inside
YDR060W. Because this fragment is located at the genetic map
position of mak21-1, YDR060W is the
MAK21 gene and not a suppressor.
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Fig. 1.
MAK21 = YDR060W. Bases
570,079-576,879 of chromosome IV were subcloned from the clone
ATCC71164 (see "Materials and Methods"). Complementation of
mak21-1 for propagation of M1 dsRNA by various
sublcones is shown above. Various parts of Mak21p were expressed and
assessed for complementation of mak21
by growth and
polysome gradients (see "Materials and Methods" and Fig. 5).
ura3/ura3 his3/his3 trp1/+
leu2/+) was replaced with the HIS3 gene using plasmid
pH75 (see "Materials and Methods" and Fig. 1), resulting in strain
YHE353, and the disruption was confirmed by Southern blotting. Of 32 meiotic tetrads from strain YHE353, 31 tetrads had only two viable
spores, and the remaining tetrad contained only one viable spore. Of
these 32 tetrads 11 were germinated at 30 °C, 10 at 25 °C, and 11 at 20 °C. Thus the germination temperature did not seem to influence
spore viability. MAK21 is thus needed for spore germination
or for growth.
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Fig. 2.
Homologs of Mak21p. Panel A,
homologous regions of Mak21p with hCBF, its mouse homolog (50), and
ORFs from S. pombe (EMBL accession number Z98980, locus
SPAC4F10, R. Connor and C. M. Churcher, unpublished data; B. G.
Barrell, M. A. Rajandream, and V. Wood, unpublished data) and C. elegans (locus CEF23B12, accession Z77659, Ref. 51). MACAW (52)
was used to determined the homologous regions. Panel B,
lineup of regions of highest homology of Mak21p and hCBF.
ura3 his3 mak21-1)
containing either pRS316 (a URA3 CEN plasmid) or pH107
(pRS316-MAK21). Transformants were grown in 50 ml of SD medium to A600 = 0.6. After resuspension of the
cells in phosphate-buffered saline, protein extracts were prepared by
disruption with glass beads. Luciferase activity was measured using the
Promega luciferase assay kit and a luminometer (10, 11) and was
corrected for the protein concentration of the extract, measured
using the Bradford assay.
leu2 trp1 ura3
mak21::HIS3 pH107 {URA3 CEN MAK21} K+)
were selected 5-fluoroorotic acid-resistant colonies. However, the
appearance of resistant cells was below 1 in 10,000, suggesting that
loss of the MAK21 plasmid was lethal. One resistant isolate
was analyzed for the presence of plasmids by Southern blotting. This
showed that recombination had taken place, resulting in loss of the
original plasmids. Thus, hCBF cannot substitute for Mak21p in
yeast.
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Fig. 3.
L-A Gag levels and L-A
dsRNA levels are reduced in a mak21-1 host. Cells were
grown in SD medium to A600 = 0.4-0.5, and
lysates were prepared in phosphate-buffered saline containing 100 µg/ml phenylmethylsulfonyl fluoride. Protein extracts were analyzed
by electrophoresis on an 8% SDS-polyacrylamide gel and blotted onto a
nitrocellulose membrane (33). L-A Gag protein was visualized using an
anti-Gag monoclonal antibody (yCP6) and anti-mouse IgG-conjugated
alkaline phosphatase (Promega). These extracts were also treated with
phenol/chloroform, and nucleic acids remaining in the aqueous phase
were ethanol precipitated. 50 µg of extracted nucleic acid was
treated with 0.01 µg/ml RNase A in the presence of 0.8 M
NaCl for 30 min at 30 °C. After phenol/chloroform extraction and
ethanol precipitation the dsRNA was dissolved in water. 10 µg of RNA
and an equivalent amount of RNase A-treated sample were analyzed by
electrophoresis on a 1% agarose gel and stained with ethidium
bromide.
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Fig. 4.
Depressed free 60 S ribosomal subunits in
mak21-1 strains. Panel A, polysome
gradients of mak21-1 and MAK21 strains are shown.
Halfmer peaks are indicated by vertical arrows. Panel
B, sucrose gradients with neither Mg2+ nor
cyclohexamide showing decreased total 60 S subunits in a
mak21-1 strain.
leu2 trp1 ura3 mak21::HIS3 K+ pH107), the
ADH1::MAK21 expression plasmid pH107
was eliminated by 5'-fluoro-orotic acid selection, making Mak21p
expression dependent on the activity of the GAL1 promoter of
pH287 (strain YHE565).
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Fig. 5.
Effects of depletion of Mak21p and deletion
mutations. Polysome gradients of YHE393
(mak21::HIS3) with the indicated plasmids were
carried out as described under "Materials and Methods." Panel
A, the normal MAK21 gene (pH183); panel D, a
GAL1::MAK21 plasmid (pH287). Both were growing on
glucose (see "Materials and Methods"). Panel B,
MAK21 S207 (pH310) and panel C,
MAK21 228 (pH311) are NH2-terminal deletions
with translation starting at residues 207 and 228, respectively, with
artificial start codons. Panel E, MAK21 898
(pH281) and panel F, MAK21 961 (pH211) have
terminators at those residues.
Reduced expression of cap
and poly(A) mRNAs due
to mak21-1
ura3 his3 mak21-1) containing either
pRS313 (HIS3 CEN) or pH110 (pRS313-MAK21) were
grown in His medium to an A600 of 0.6. Cells were treated
with lyticase and electroporated with one of four different luciferase
mRNAs. The luciferase RNAs were capped and polyadenylated (C+A+),
only capped (C+A), only polyadenylated (CA+), or contained neither
a cap nor a poly(A) tail (CA). Four independent experiments were
performed, and luciferase production was measured 2 h after
electroporation. Each data point is the average of two
electroporations. The same batch of RNA was used in Experiments 1 and 2 and a new batch for Experiments 3 and 4. Luciferase activity is
expressed in arbitrary units corrected for the protein content of the
extracts. Protoplast preparation, electroporation, and luciferase
assays were performed as described (11).
mak21-1 reduces expression of CA L-A-luciferase hybrid mRNAs
A, and experimental
details were as in Table I.
leu2 trp1 ura3
mak21::HIS3 pH107 {URA3 CEN MAK21} K+).
Complementation of MAK21 function was analyzed by spotting 10,000 cells
onto a 5'-fluoro-orotic acid plate (thereby selecting for loss of the
plasmid carrying the normal MAK21 gene) and scoring for
growth.
mutation (YHE393), both for growth
and ability to maintain M1, indicating that it had fully
retained Mak21p activity. The GFP-Mak21 fusion protein localized to a
discrete spot in the yeast cells (Fig. 6). However, formaldehyde fixation and/or
digitonin permeabilization of these cells (for the purpose of staining
the nuclei with DAPI) resulted in a diffuse distribution of the
fusion protein, precluding the ability to relate GFP-Mak21p
localization to specific cellular domains. A fusion protein from which
the COOH-terminal amino acids were deleted up to Met-827 (pH283) was no
longer able to complement a mak21 mutation. This fusion
protein also lost the discrete cellular localization of the full-length
fusion protein (Fig. 6).
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Fig. 6.
Localization of Mak21p-GFP fusion
protein. Top panels, phase-contrast micrographs.
Lower panels, fluorescence micrographs (GFP).
Left, YHE393 (mak21::HIS3 pH107
(MAK21)) carrying pH209, a CEN plasmid in which the
5'-GFP-MAK21 3' fusion protein is expressed from the MAK21
promoter. A similar localization was observed when this strain lacked
pH107. Center, YHE393 carrying pH283, with M827 changed to a
stop codon. Right, GFP expressed alone from the
MAK21 promoter in YHE393.
Suppress mak21-1 Effects on M1
Maintenance but Not on 60 S Levels--
In general the effect on
M1 satellite RNA propagation of mak mutations is
suppressed by ski mutations. This is probably also true for
mak21-1 because this mutation is suppressed by
ski4 (40). However, the effect of mak mutations,
ski mutations, or the presence of both mak and
ski mutations on L-A propagation in the absence of M has not
been analyzed in detail. To do so, mak21-1 ski6-2 and
mak21-1 ski2 double mutants were constructed. These
strains maintained M1, showing that indeed the
mak21-1 mutation can be suppressed by ski
mutations. Introduction of SKI6 or SKI2
expression plasmids into the corresponding mak21-1 ski6-2 or
mak21-1 ski2 strains resulted in the loss of
M1. Allowing the loss of these plasmids produced mak
ski double mutants in which L-A propagation was not influenced by
M1. After subsequently introducing MAK21, SKI6,
or SKI2 expression plasmids, or just the expression vectors into these strains, the effect of mak21-1 and
ski6-2, or ski2 mutations on L-A
propagation was studied. L-A Gag levels were modestly decreased in
mak21-1 mutants compared with the wild-type cells, and L-A
levels were clearly increased in ski6-2 or
ski2 mutants. In mak21-1 ski6-2 or
mak21-1 ski2 double mutants L-A levels at least equaled
those found in cells having only the ski mutation (data not
shown).
mutation on polysome profiles
was analyzed. We showed above that mak21-1 causes a decrease
in 60 S ribosomal subunits resulting in the appearance of halfmer
polysomes. Although ski6-2 strains accumulate an abnormal
60 S subunit sedimenting at 38 S (11), this was not prominent on our
gradients done on cells grown at a permissive temperature (30 °C).
The ski2 strain also had a normal polysome profile. When
a mak21-1 mutation is combined with either a
ski6-2 or a ski2 mutation the polysome
profiles of the resulting strains are similar to those of strains
containing only the mak21-1 mutation (Fig.
7).
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Fig. 7.
ski mutations suppress virus loss
phenotype of mak21-1 but not 60 S deficiency.
Polysome profiles were prepared from strains YHE414
(mak21-1), YHE561 (ski6-2),YHE626
(ski2 ), YHE618 (mak21-1 ski6-2), and YHE633
(mak21-1 ski2). Cells were grown in YPAD to an
A600 of approximately 0.5.
DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
mRNAs, as we observe. However, another model
suggests that poly(A) is involved in attracting 40 S initiation
complexes to the mRNA (48). This model would predict that some of
the mak mutants would be deficient in 40 S subunits, a
result we have not found (49). Only one of several 40 S-deficient
strains loses M1 dsRNA, suggesting that 40 S deficiency
per se is not preferentially disadvantageous to non-poly(A)
mRNAs in vivo. It is not clear by either model why
deficiency of 60 S subunits would put cap-deficient mRNAs at more
of a disadvantage than they are already. Further work will be necessary
to resolve this problem.
ACKNOWLEDGEMENTS
FOOTNOTES
Present address: Central Research Laboratory, Asahi Breweries,
Ltd., 2-13-1, Ohmori-kite, Ohta-ku, Tokyo 143 Japan.
REFERENCES
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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