(Received for publication, October 7, 1996, and in revised form, March 22, 1997)
From the Department of Biological Sciences, The translation product of the VMA1
gene of Saccharomyces cerevisiae undergoes protein
splicing, in which the intervening region is autocatalytically excised
and the franking regions are ligated. The splicing reaction is
catalyzed essentially by the in-frame insert, VMA1-derived
endonuclease (VDE), which is a site-specific endonuclease to mediate
gene homing. Previous mutational analysis of the splicing reaction has
been concentrated extensively upon the splice junctions. However, it
still remains unknown which amino acid residues are crucial for the
splicing reaction within the entire region of VDE and its neighboring
elements. In this work, a polymerase chain reaction-based random
mutagenesis strategy was used to identify such residues throughout the
overall intervening sequence of the VMA1 gene.
Splicing-defective mutant proteins were initially screened using a
bacterial expression system and then analyzed further in yeast cells.
Mutations were mapped at the N- and C-terminal splice junctions and
around the N-terminal one-third of VDE. We identified four potent
mutants that yielded aberrant products with molecular masses of 200, 90, and 80 kDa. We suggest that the conserved His362, newly
identified as the essential residue for the splicing reaction, contributes to the first cleavage at the N-terminal junction, whereas
His736 assists the second cleavage by Asn cyclization at
the C-terminal junction. Mutations in these regions did not appear to
destroy the endonuclease activity of VDE.
Protein splicing is a posttranslational process, in which an
intervening protein sequence is autocatalytically removed from a
precursor protein and the two flanking sequences are ligated (1). In
the budding yeast Saccharomyces cerevisiae, the nascent 120-kDa VMA1 translational product (Vma1 protozyme (1))
catalyzes protein splicing to yield a 70-kDa catalytic subunit of the
vacuolar H+-ATPase and a 50-kDa site-specific endonuclease
(VMA1- derived
endonuclease; VDE)1 (2, 3).
Since the discovery of protein splicing in S. cerevisiae (2,
3), this compelling reaction has been found in a number of protozymes
in eucarya (2-4), bacteria (5-8), and archaea (9, 10).
The VDE region in the Vma1 protozyme plays a central role for the
protein splicing reaction (11, 12). We have also shown that VDE
bracketed by only 6 proximal and 4 distal amino acids is
autocatalytically processed in vitro (13). These results support the idea that the VDE sequence and a few external amino acids2 contain sufficient information for
protein splicing.
Only short amino acid sequence motifs are conserved among protein
splicing elements (7). Thiol- or hydroxyl-containing Cys, Ser, or Thr
is found at both splice junctions. A few hydrophobic amino acids are
present in front of an invariant His-Asn dipeptide at the C-terminal
splice junction. These residues around the splice sites have been found
to play key roles in the protein splicing reaction, based mostly on
site-directed mutagenesis studies (5, 10, 11, 12).
To understand a mechanism and structural integrity for protein splicing
in the Vma1 protozyme, we performed a systematic search for
splicing-defective mutants. We introduced random mutations throughout
the entire VDE region by an error-prone PCR method for the first time
and mapped three core regions essential for the splicing reaction.
Strains To express a GST-Vma1 fusion protein, an
expression plasmid pGEX-VMA1 was created as follows. A 652-bp
ScaI-ScaI fragment from plasmid pMVMA1 (11) was
ligated into the vector pGEX-5X-3 (Pharmacia Biotech Inc.), which had
been digested with EcoRI and blunt-ended by T4 DNA
polymerase. The resultant plasmid, which had the insert in the correct
orientation, was digested with BglII and SmaI,
and then a 1.8-kb BglII-NcoI (blunted) fragment
from pMVMA1 was introduced into this gap to generate plasmid pGEX-VMA1. The numbering of the amino acid sequence in this report refers to the
numbering of the original VMA1 gene product (2).
To express the full-length VMA1 gene product in yeast cells,
the 2.2-kb KpnI-SphI fragment of the pGEX-VMA1
plasmid was ligated into pSN001 that had been digested with the same
enzymes.
To express recombinant VDEs in E. coli, a plasmid pET-17bVDE
was constructed as follows. The pET-17b vector (Novagen) was digested
with NheI and BamHI, blunted with T4 DNA
polymerase, and self-ligated. The resultant plasmid was digested with
EcoRV, and a MluI linker, dGACGCGTC, (New England
Biolabs) was inserted into this gap to create a stop codon, yielding
pET-17b VDE cleaves the VMA1 gene, which lacks the VDE coding
sequence (VMA1 Random mutations were introduced throughout
the VDE region of pGEX-VMA1 plasmid by error-prone PCR (16). The
5 E. coli strain SCS1 carrying mutagenized
pGEX-VMA1 was cultured in 1 ml of LB medium containing 100 µg/ml
ampicillin in 24-well tissue culture plates. The culture was grown at
37 °C for about 6 h to the exponential phase. Then 0.1 mM IPTG was added, and the culture was transferred to room
temperature. After an additional 3-h incubation, total cell extracts
were prepared by pelleting the cells for 2 min at 10,000 × g followed by lysis in 50 µl of SDS-PAGE sampling buffer
(62.5 mM Tris-HCl, pH 6.8, 2% SDS, 1% 2-mercaptoethanol,
10% glycerol, 0.01% bromphenol blue) and boiling for 3 min. Samples
(5 µl) were directly analyzed by 8% SDS-PAGE, and then protein was
visualized by Coomassie Brilliant Blue staining.
Mutants could be classified into roughly five groups. One group of
mutants (100 mutants out of the 324 screened mutants) exhibited the
excised 50-kDa VDE predominantly near wild-type levels. A second group
of mutants (106 mutants) showed a somewhat reduced level of processing,
accumulating a comparable amount of the 50-kDa VDE and the 115-kDa
precursor. A third group of mutants (33 mutants) exhibited a markedly
reduced amount of the 50-kDa VDE and predominant accumulation of the
precursor. A fourth group of mutants (44 mutants) failed to show any
detectable 50-kDa VDE in a Coomassie Blue-stained gel. A fifth group of
mutants (41 mutants) failed to express the full-length precursor,
presumably because a stop codon was introduced into the open reading
frame.
Mutants of the fourth group identified in the initial screen were then
subjected to Western blotting analysis, which was done essentially as
described previously (2). Then 17 mutants were found not to produce any
immunodetectable splicing products. DNA sequencing of these mutant
plasmids was done by using the Sequenase kit (U.S. Biochemical Corp.)
with appropriate primers. The number of amino acid substitutions of
each mutant varied from one to six. In addition, mutants 93, 95, 175, 198, 323, and 330 contained a single silent mutation, and mutant 347 had two silent mutations. Then individual mutant plasmid DNAs were
dissected by restriction fragment swapping to identify which amino acid
changes were responsible for the defect of splicing activity. The
VDE-coding region was divided into three intervals: segment 1 (530-bp
BamHI-BglII), segment 2 (760-bp
BglII-SacII), and segment 3 (150-bp
SacII-EcoRI). These segments of each mutant DNA
were individually ligated into wild-type pGEX-VMA1 plasmid to replace
the corresponding regions. As for the mutant 330, which had a mutation
upstream of the KpnI site, DNA was dissected to segment 0 (60-bp BamHI-KpnI) and segment 1 Yeast strain NY101
carrying pSN001 plasmid was used for the complementation test of mutant
VMA1 genes. pYO314 plasmid was used as a control
vector.2 The complementation test was done using YPD medium
(1% yeast extract, 2% polypeptone, 2% glucose) supplemented with 100 mM CaCl2. Wild-type and mutant cell extracts
were prepared from cultures grown at 30 °C in YNBD medium (0.67%
yeast nitrogen base without amino acids and 2% glucose) supplemented
with 0.5% casamino acids and buffered with 50 mM
succinate/NaOH, pH 5.0. Preparation of cell extract and Western
blotting analysis was done as described previously (2, 13).
E.
coli BL21(DE3) carrying plasmid pET-17bVDE was cultured in LB
medium containing 100 µg/ml ampicillin. A 0.1-ml overnight culture
was added to 5 ml of fresh medium and grown at 23 °C to the early
exponential phase (A600 = 0.07). Protein
expression was then induced by the addition of 0.1 mM IPTG
for 4 h to an A600 of about 0.2. Cells were
pelleted and resuspended in 1 ml of lysis buffer (50 mM
Tris-HCl, pH 8.0, 2 mM EDTA, 20 mM
MgCl2) containing 1 mg/ml lysozyme (Sigma). After
incubation on ice for 30 min, cells were lysed by three freeze-thaw
cycles. The subsequent purification steps were carried out at 4 °C.
The lysates were clarified by centrifugation at 10,000 × g for 10 min. Solid ammonium sulfate was added to the
supernatant up to 70% saturation. The precipitate was collected by
centrifugation (10,000 × g for 10 min) and resuspended
in 1 ml of buffer A (10 mM sodium phosphate, pH 7.4, and
0.1 mM EDTA). The resuspended solution was dialyzed against
buffer A for 2 h. The dialysate was batch-loaded onto 0.2 ml of
CM-Sepharose CL-6B beads (Pharmacia) previously equilibrated with
buffer A. The mixture was rotated for 10 min, and beads were pelleted
and washed with 1 ml of buffer A. Bound proteins were then eluted with
0.5 ml of buffer A containing 200 mM NaCl. This fraction
was dialyzed against buffer B (10 mM Tris-HCl, pH 8.0, and
0.1 mM EDTA) and used for endonuclease activity analysis. The protein concentration of the sample was estimated using BCA reagent
(Pierce).
Endonuclease activity was assayed according to a modification of the
method of Gimble and Stephens (17) using pBS- A bacterial
expression system was used to produce normal and mutagenized
recombinant Vma1 protozymes for monitoring their processing activity.
The sequence, which covers the entire VDE region and the C-terminal
segment of the VMA1 gene, was fused to the GST gene under
control of the tac promoter (Fig.
1A). This construct expresses a chimeric
protein (GST-Vma1) that is composed of GST and 11 residues from the
Vma1 N-terminal region (~30 kDa), VDE (50 kDa), and the Vma1
C-terminal region (35 kDa). When this authentic GST-Vma1 fusion protein
was expressed in E. coli, a 115-kDa precursor was processed
into a 50-kDa VDE and a 65-kDa GST-Vma1 C-terminal fusion protein. The
115-kDa precursor and the excised 50-kDa VDE were easily detected by
Coomassie Blue staining (Fig. 1B) and also by anti-VDE
polyclonal antibody (Fig. 1C). The 65-kDa spliced product
(GST-Vma1C) was hardly detected by Coomassie Blue staining, but it was
detected by 5M39 monoclonal antibody (Fig. 1D), which
recognizes the C-terminal region of Vma1p (11). We found that protein
splicing efficiency in E. coli was temperature-sensitive.
Induction at 37 °C resulted in predominant accumulation of the
115-kDa precursor in an insoluble fraction (data not shown). Induction
at lower temperatures yielded a larger amount of the processed 50-kDa
VDE, which was recovered in a soluble fraction.
Mutations were introduced randomly throughout the entire VDE
region of the GST-VMA1 fusion gene by error-prone PCR. The
mutagenized 1.4-kb BamHI-EcoRI fragment covers
the entire VDE and external 14 proximal and 13 distal amino acids.
After induction at room temperature with 0.1 mM IPTG, total
proteins of E. coli cell lysate were examined by SDS-PAGE
followed by Coomassie Blue staining. The splicing activity of mutants
was estimated by comparing the density of the 115-kDa precursor band
with the density of the 50-kDa band of excised VDE. Defective mutants
(44 mutants of the 324 candidate mutants) that accumulated the
precursor and did not show detectable splicing products in the
Coomassie Blue-stained gels were then subjected to the second level of
screening using Western blotting analysis. Of the 44 mutants tested, 17 mutants showed no immunodetectable amount of the excised 50-kDa VDE
against the anti-VDE antibody. The plasmid DNAs from these 17 mutants were purified and sequenced within the mutagenized region. The results
of the sequencing are shown in Table I.
Table I.
Mutations that abolish protein splicing of GST-Vma1p in E. coli
Faculty of Pharmaceutical Sciences,
University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, Japan
Escherichia coli strain SCS1 carrying
plasmid pGEX-VMA1 (see below) was used to produce the recombinant
GST-Vma1 fusion protein. E. coli strain BL21(DE3) carrying
plasmid pET-17bVDE (see below) was used to produce recombinant VDE.
Yeast strain NY101,2 which is a
vma1::URA3 derivative of YPH499,
containing plasmid pSN001,2 was used to express the
full-length VMA1 gene product.
NE. The BamHI-EcoRI fragment of
wild-type or mutant pGEX-VMA1 was introduced into the
BamHI-EcoRI gap of pET-17b
NE to create
pET-17bVDE. The pET-17bVDE expresses VDE bracketed by 18 proximal
(MARIPRNYSNSDAIIYVG) and 17 distal (CGERGNEMAEVLMEFCR) amino acids.
vde) at the precise insertion
site (14, 15). A plasmid substrate pBS-
VDE, which contains a VDE
cleavage site, was constructed as follows. The 1.7-kb
PvuII-EcoRI fragment of pMVMA1 was inserted into
Bluescript SK+ (Stratagene), which had been digested with
SmaI and EcoRI, to produce pBS-PE. A VDE cleavage
site was generated by a PCR-based deletion mutagenesis using a standard
PCR condition and Pfu polymerase (Stratagene). One reaction
mixture contained oligonucleotide primer A
(5
-TTGCTCCAGCTGGTGAGTACA-3
) (codons 557-577); primer B
(5
-ACCTCTTTCTCCGCACCCGACATAGATAATGGCGTCA-3
), which has 15 nucleotides (codons 2212-2226) followed by 22 nucleotides (codons
828-849) of the antisense strand; and pMVMA1 as a template. Another
reaction mixture contained primer C
(5
-ATCTATGTCGGGTGCGGAGAAAGAGGTAATGAAATG-3
), which has 12 nucleotides
(codons 838-849) followed by 24 nucleotides (codons 2212-2235) of the
sense strand; primer D (5
-TCTCAAAGCTTCAGCCCATCT-3
) (codons
2440-2460); and pMVMA1. The two PCR products were mixed, and the first
denaturation and annealing reaction was performed. Then polymerase and
primers A and D were added, and elongation and additional cycles were
carried out. The PCR product was digested with ScaI and
EcoRI, and the resultant 72-bp
ScaI-EcoRI fragment was gel-purified and
introduced into the 1.4-kb ScaI-EcoRI gap of
pBS-PE to yield pBS-
VDE. The ScaI-EcoRI region
of pBS-
VDE was confirmed by direct sequencing.
-primer was 5
-CGTGGGATCCCCAGGAATTACTCCAATTCT-3
,
which contains a BamHI site (underlined). The
3
-primer was 5
-TGGGAATTCCATCAAGACTTCTGCCATTTC-3
, which
contains an EcoRI site (underlined). The reaction mixture contained 100 ng of pGEX-VMA1 plasmid, 0.01 OD units of each primer, 0.2 mM dGTP, 0.2 mM dATP, 1 mM
dCTP, 1 mM dTTP, 50 mM KCl, 10 mM
Tris-HCl, pH 8.3, 0.01% gelatin, 7 mM MgCl2,
and 5 units of Taq polymerase (Takara) in a 100-µl volume.
To avoid extreme mutagenic effect, MnCl2 was not
supplemented. The reaction conditions were as follows: 25 cycles of
94 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min. The PCR
product was digested with BamHI and EcoRI and
then ligated to pGEX-VMA1 that had been digested with the same
enzymes.
(470-bp
KpnI-BglII). Mutant 197 could not be dissected,
because the mutation abolished the BglII restriction site.
Then splicing activities of the reconstituted mutants (designated by
the addition of hyphened segment numbers) were reexamined by
immunoblotting. Nineteen reconstituted mutants did not show any
detectable amount of the 50-kDa VDE (among them, the mutant 239-1 showed a very slight amount of the 50-kDa VDE near the background level
of detection). The other mutants with splicing ability were
excluded.
VDE as a substrate.
Purified recombinant VDE (0.5 µg) was incubated with XmnI-linearized plasmid pBS-
VDE (600 ng) in 15 µl of
cleavage buffer (20 mM Tris-HCl, pH 8.5, 10 mM
MgCl2, 100 mM KCl, and 1 mM
dithiothreitol) for 1 h at 37 °C. Five µl of the reaction
mixture was electrophoresed in a 0.7% agarose gel with 0.5 × TAE
(Tris acetate-EDTA) buffer and visualized by ethidium bromide
staining.
Expression of GST-Vma1 Fusion Protein in E. coli
Fig. 1.
Construction and expression of wild-type and
mutant GST-Vma1 fusion proteins in E. coli. A,
the region that covers VDE and the C-terminal segment of the
VMA1 gene was fused to the GST gene. E. coli cell
extracts that expressed wild-type (lane 1) or mutagenized
GST-Vma1 fusion proteins (lanes 2, 3, and
4) were analyzed by 8% SDS-PAGE. B, Coomassie
Brilliant Blue staining. C, Western blotting with anti-VDE
polyclonal antibody. D, Western blotting with 5M39
monoclonal antibody. In wild-type (WT) cells, a 115-kDa
precursor was processed into the 50-kDa VDE and the 65-kDa GST-Vma1
C-terminal fusion protein.
[View Larger Version of this Image (59K GIF file)]
Mutant No.
No. of amino acid
changes
BamHI-KpnI
KpnI-BglII
BglII-SacII
SacII-EcoRI
89
4
K539R,N594Y
H725L,N737I
93
3
M311K,N434T
N737S
95
2
R325S
N737K
115
3
M330K,T361I,M392T
129
3
A463T,D607G,A648V
175
2
T357S,H362L
197
6
K322E,V365A
D446V,aG451V,I485F,E524V
198
5
D277G,N303T,R325S
V644G,A682T
238
3
V291G,M293T
Y475H
239
3
T357S,D399V
Y664N
265
1
C284Y
282
5
N290K,I298F
L449P,Q460L
E740A
303
3
H362L
L640I,T658A
323
2
V403D
S639P
327
3
K478N
D724V,L729P
330
2
G283V
T368A
347
2
K552R,L602H
a
The D446V mutation of mutant 197 abolished the
BglII restriction site.
Mutations were distributed throughout the entire mutagenized region,
and the number of amino acid substitutions in each mutant varied from
one to six. To identify which amino acid changes are responsible for
the defect of splicing activity, individual mutant plasmid DNAs were
dissected by restriction fragment swapping (see "Experimental
Procedures"). Then the splicing activity of the reconstituted mutants
was reexamined by immunoblotting. Nineteen reconstituted mutants did
not yield any detectable 50-kDa VDE. These mutations were distributed
in their sequences as shown in Fig. 2. Of the 19 plasmids, 7 plasmids contained a single amino acid substitution. Two of
them (G283V and C284Y) were at the N-terminal splice junction, and
another two (N737S and N737K) were at the C-terminal junction. These
four mutations at the splice junctions are in agreement with previous
studies (5, 10, 11, 12). We found three additional new mutants within
the VDE region (H362L, V403D, and S639P). As for the conserved
His362 (7), it was substituted in two mutants (175-1 and
303-1).
Protein Splicing Activity of Mutant VDEs in Yeast
We next
tested whether these mutants defective in splicing in E. coli show the defect in yeast cells as well. The mutant VDE genes
were introduced into a plasmid carrying the entire VMA1 gene
to replace the wild-type VDE region. The splicing activity of mutant
Vma1 protozymes was tested by their ability to complement the
calcium-sensitive growth defect of vma1 cells. A loss of protein splicing of the Vma1 protozyme leads to a lack of Vma1p, the
70-kDa catalytic subunit of the vacuolar H+-ATPase. Since
yeast mutants that lack Vma1p are sensitive to calcium-containing
medium (18), the ability to complement calcium sensitivity correlates
with the splicing activity in yeast (11). We found that several mutants
were capable of complementing the calcium-sensitive growth defect of
vma1 strain fully or partially (Fig. 3 and
Table II), suggesting that the efficiency of protein splicing may be different in yeast and E. coli cells.
|
Of 17 mutants tested, six mutants (89-3, 93-3, 95-3, 175-1, 238-1, and
327-3) could not complement calcium sensitivity at all and also did not
yield correctly excised products in yeast cell lysates (Figs. 2 and
4). Thus, we concluded that these six mutants had lost
the splicing activity completely.
Western blotting analysis with anti-VDE antibody was performed on yeast cell lysates from the mutant strains. The excised 50-kDa VDE was detected in the cell lysates in response to their ability to grow on the calcium-containing medium, showing that protein splicing occurred normally in some strains (Fig. 4A). Interestingly, we could detect several protein species with intermediate sizes between the 120-kDa precursor and excised 50-kDa VDE in four mutants 89-3, 95-3, 175-1, and 303-1. These species were further characterized by their sizes and reactivity to the antibodies raised against the three regions of the Vma1 protozyme (Fig. 4B); R70 (monoclonal antibody for the 30-kDa N-terminal region), anti-VDE polyclonal antibody, and 5M39 (monoclonal antibody for the 40-kDa C-terminal region; Refs. 11 and 13). In all four mutants, a 120-kDa precursor polypeptide accumulated, which was recognized clearly by the three antibodies (Fig. 4B). Anti-VDE and 5M39 antibodies reacted with a 90-kDa polypeptide accumulated in the mutants 89-3 (H725L and N737I) and 95-3 (N737K), suggesting that the cleavage occurred only at the N-terminal splice site. The mutant 95-3 also accumulated a more slowly migrating polypeptide around 200 kDa that was recognized by all the three antibodies, suggesting that it could be a branched protein (19). We are not certain about the origin of a 54-kDa band in the mutants 89-3 and 95-3, which was detected only by anti-VDE antibody. As for mutants 175-1 (T357S and H362L) and 303-1 (H362L), an 80-kDa polypeptide was detected with anti-VDE and R70 antibodies, suggesting that the cleavage occurred only at the C-terminal splice site. These observations obtained with new VDE mutants are important for considering the mechanism of protein splicing (see "Discussion").
Endonuclease Activity of the Splicing-defective VDE MutantsWe then examined whether the defect in protein splicing
affects the endonuclease activity of VDE. To produce the wild-type VDE
and the splicing-defective VDE in E. coli, the
BamHI-EcoRI fragment was connected downstream of
the T7 promoter. The resulting construct was expected to express a VDE
polypeptide bracketed by 18 proximal and 17 distal amino acids (Fig.
5A). When the wild-type polypeptide was
expressed at 23 °C, two protein bands were observed around 50 kDa
(Fig. 5B). It is plausible that the minor upper band
represents the precursor and that the major lower band represents the
excised VDE, since the upper band disappeared after purification (13).
None of the mutants produced the excised form, consistent with their
inability to splice the external regions. However, protein products of
intermediate sizes were seen in several mutants on an SDS-PAGE
gel (Fig. 5B), probably resulting from aberrant processing
or proteolysis, as observed in yeast cell lysates (Fig. 4).
The majority of the products were recovered in a soluble fraction of
E. coli lysate, with the exception of mutants 238-1 and 327-3 (Fig. 5B). These two mutant proteins were highly
insoluble and could not be purified. Other mutant VDE proteins could be easily purified by ammonium sulfate precipitation and CM-Sepharose CL-6B chromatography, although heterogeneous polypeptides were copurified together in some cases (Fig. 5B). The
endonuclease activity of mutant VDEs was assayed with these samples
using a plasmid containing the specific sequence
(VMA1vde) as substrate. Then we found that all
of the purified mutant proteins had normal cleavage activity (Fig.
5C). As control, we used a bracketed VDE mutant, 64-2, which
contains two mutations, D609V and V643A. The residue Asp609
is in the dodecapeptide motif and known to be essential for
endonuclease activity (17). This mutant showed normal splicing activity
(Fig. 5B), but could not cleave the substrate DNA (Fig.
5C).
In this study, we discovered that the His362 residue of the Vma1 protozyme is critical for protein splicing, in addition to the previously mentioned residues around the N- and C-terminal splice junctions (5, 10-12). We noticed that the induction at a high temperature (37 °C) decreased the splicing efficiency in E. coli. A lower temperature (~25 °C) seemed to allow productive folding of the precursor for splicing. Interestingly, several mutants that could not splice in E. coli cells were found to splice in yeast cells to some extent. This may be due to differences in conditions of the two cell systems, such as the rate of precursor synthesis, intracellular pH, ion composition, reducing potentials, and molecular chaperones.
We analyzed six potent mutants in detail, which showed abortive splicing in yeast cells and completely lost normal splicing activity. The amino acid residues altered in these six mutants were found to cluster in three core regions. They were located around the N-terminal splice junction (mutant 238-1), the C-terminal splice junction (mutants 89-3, 93-3, 95-3, and 327-3), and the N-terminal one-sixth portion of VDE (mutant 175-1).
N-terminal Splice JunctionMutant 238-1 had two mutations, V291G and M293T. Cooper et al. (12) previously reported that the changes of the Val291 residue (V291F and V291D) completely blocked splicing in yeast. Although mutants 265-1 (C284Y) and 330-0 (G283V) were not tested in yeast cells, these are already known to be essential (11, 12).3 To our knowledge, the G283V mutation is the first example (except for the Cys738 residue at the C-terminal splice junction) showing an essential requirement of the residue outside the VDE region for protein splicing. In addition, Nogami et al. have recently demonstrated that the three hydrophobic residues (Ile279-Ile280-Tyr281) upstream of the N-terminal junction genetically interact with the hydrophobic residues (Val733-Val734-Val735) at the C-terminal junction to fulfill the splicing reaction.2 Our current results suggest that the Gly283 residue at the N-terminal junction may serve the splicing reaction by conferring conformational flexibility to the splice site, although Cooper et al. (12) reported that the G283R mutation did not affect the splicing efficiency.
C-terminal Splice JunctionAsn737 at the C-terminal junction plays a critical role in protein splicing to form a succinimide ring (20). Cooper et al. (12) reported that substitutions of Asn737 to seven other amino acids (Lys, Ala, Tyr, Gln, Glu, His, and Asp) all resulted in nonspliced products. In this study, we obtained three mutants in which the Asn737 residue was substituted (mutants 89-3, 93-3, and 95-3), in good agreement with the previous observations (11, 12). Mutant 327-3 had two mutations near the C-terminal junction (D724V and L729P). We found that mutant 89-3 (H725I and N737I) and 95-3 (N737K) accumulated in yeast cells a 90-kDa side product that is supposed to result from cleavage at the N-terminal splice site (Fig. 4B). Hirata and Anraku (1, 11) also showed previously that N737V mutant accumulates a 90-kDa product. Furthermore, mutant 95-3 (N737K) accumulated a species that migrated more slowly (around 200 kDa) than the 120-kDa precursor and reacted with three antibodies (Fig. 4B). Thus, this species is likely to be a branched molecule that had been observed in an in vitro splicing reaction of the precursor from Pyrococcus DNA polymerase (19). Recently, Chong et al. (21) also reported that a branched intermediate accumulated in vivo using the N737A/C738S mutant of VDE. Our results are consistent with the succinimide-mediated cleavage model at the C-terminal junction (20).
The Region around His362The third region crucial for protein splicing was newly identified in this study. The substitution of His362 with Leu caused a serious defect in protein splicing in yeast (mutant 303-1). An additional conservative mutation in the neighborhood (T357S) blocked splicing completely (mutant 175-1). These mutants yielded a 80-kDa C-terminal cleavage product (Fig. 4B). This suggests that His362 is required for the first cleavage at the N-terminal splice site.
Chong et al. (21) investigated the side reaction products accumulated in the VDE mutants that substituted the four key residues at the splice junctions: Cys284, His736, Asn737, and Cys738. They proposed that protein splicing of the yeast Vma1 protozyme occurs via a mechanism similar to that in the thermophilic archaea (20, 21). According to their model, splicing proceeds as follows: step 1, N-S acyl rearrangement at the N-terminal splice junction involving Cys284; step 2, transesterification involving Cys738 to yield a branched intermediate; step 3, peptide cleavage at the C terminus by cyclization of Asn737; and step 4, S-N acyl shift of the transient spliced product to yield a normal peptide bond. In our study, the H362L mutant allowed cleavage at the C-terminal splice site, while it prevented the N-terminal scission. Thus, we suggest that the H362L mutant is likely to have a defect in step 1 or 2. The His362 residue may assist the N-S rearrangement of the Cys284 residue (step 1) or be required to activate the Cys738 residue as a nucleophile for the transesterification reaction (step 2) by acting as a proton acceptor for the thiol of either Cys residue.
Pietrokovski (7) pointed out that the His362 residue is invariable among all of the known protein splicing elements (His in motif B). Our results provide the first experimental evidence for the crucial requirement of the His362 residue. Besides the dodecapeptide motifs, there are only three invariable residues among protein splicing elements; in the Vma1 protozyme they are the His736-Asn737 dipeptide at the C-terminal splice junction and His362 (7). Like a H736L mutant (12), the H362L mutant does not block splicing completely and allowed a low level of splicing activity (Table II). Chong et al. (21) reported that a His736 mutant (H736L/C738S) as well as Asn737 mutants accumulated the branched intermediate. This suggests that His736 participates in cyclization of Asn737 to resolve the branched intermediate, which is the late step of the splicing reaction. Conversely, His362 may participate in the early step of the reaction.
Endonuclease Activity of Nonsplicing Mutant VDEsThe present
work also demonstrates the independence of two functions of VDE:
protein splicing activity and endonuclease activity. VDE has been shown
to be a site-specific endonuclease (14). Cleavage of the
VMA1vde gene by VDE in a
VMA1/VMA1
vde heterozygote initiates
gene homing that converts the VMA1
vde allele
into VMA1 (14). The dodecapeptide motifs in VDE are crucial
for the cleavage activity (17). The mutagenesis of the dodecapeptide
motif in I-TliI abolished endonuclease activity, whereas
protein splicing was unaffected, indicating that the endonuclease
activity is not required for splicing (10). In this study, the
splicing-defective VDE mutants were expressed in E. coli and
purified to assess semiquantitatively their endonuclease activities
in vitro. Then all mutants tested (89-3, 93-3, 95-3, 175-1, 265-1, and 330-0) appeared to possess endonuclease activities near the
wild-type level. These results also confirm that these mutations do not
cause global conformational changes of the protein.
In summary, our findings suggest that the catalytic site(s) for protein splicing of the Vma1 protozyme can be divided into at least three core regions in the primary structure: the N- and C-terminal splice junctions and the region around the His362 residue. This His362 residue may contribute to the N-terminal cleavage step in the splicing reaction. Furthermore, mutations in these regions do not abolish the endonuclease activity of VDE.