(Received for publication, September 8, 1994; and in revised form, December 9, 1994)
From the
Translation initiation factor Prt1 was purified from a ribosomal salt wash fraction of Saccharomyces cerevisiae cells by ammonium sulfate precipitation, DEAE chromatography, phosphocellulose chromatography, sucrose density gradient centrifugation, and non-denaturing polyacrylamide gel electrophoresis. Prt1 protein cofractionates with four other polypeptides during all steps of purification suggesting that it is part of a protein complex containing polypeptide subunits with apparent molecular masses of 130, 80, 75 (Prt1), 40, and 32 kDa. Deletion of the first AUG codon in the published sequence of the PRT1 gene results in the synthesis of functional Prt1 protein indicating that the actual molecular mass of the Prt1 subunit is 82.7 kDa. This is in agreement with results from primer extension experiments reported earlier by Keierleber et al. (Keierleber, C., Wittekind, M., Qin, S., and McLaughlin, C. S.(1986) Mol. Cell. Biol. 6, 4419-4424). The Prt1-containing protein complex is an active translation factor as shown by its ability to restore translation in a cell-free system derived from a temperature-sensitive prt1 mutant strain in which endogenous Prt1 activity is inactivated by heating the extract to 37 °C. The question of whether the Prt1-containing protein complex represents the yeast homologue of mammalian translation initiation factor eIF-3 is discussed.
Translation initiation is a multistep pathway, which positions
an 80 S ribosome with bound initiator Met-tRNA at the
initiator AUG codon of an open reading frame on
mRNA(1, 2) . In eukaryotes, the main initiation
pathway is cap-dependent; ribosomes bind near the cap structure
m
GpppN at the 5` end of mRNA and then scan the mRNA in the
5` to 3` direction until they encounter an initiator AUG
codon(3) . These reactions are catalyzed by a large number of
polypeptides, the eukaryotic initiation factors
(eIFs)(
)(1, 2) .
Experiments with the yeast Saccharomyces cerevisiae revealed that translation initiation in this lower eukaryote strongly resembles cap-dependent initiation in mammals(4, 5, 6) . This is perhaps most convincingly demonstrated by the finding that some mammalian initiation factors can substitute for yeast factors in vivo(7, 8) . The availability of yeast cell-free translation systems (9, 10) and powerful genetic approaches make this system an attractive model system to study the mechanism and regulation of translation in eukaryotes.
Most of the translation initiation factors identified earlier in the mammalian system have also been isolated and their genes cloned from S. cerevisiae(4, 5, 6) . In addition, yeast initiation factors were identified and their genes cloned whose mammalian homologues are not yet known(6, 11) .
Among the few initiation factors that have not yet been isolated
from yeast is the factor eIF-3. Mammalian eIF-3 is composed of eight
polypeptide chains with a total mass of about 550
kDa(1, 2) . Reconstitution experiments showed that
this factor is involved in several consecutive steps in the initiation
pathway including ribosome dissociation, Met-tRNA binding
to 40 S ribosomes, and mRNA binding to 40 S
Met-tRNA
complexes(1) .
The analysis of the yeast mutant strain prt1-1 originally isolated by Hartwell (12) showed that Prt1 protein stimulates the interaction of the
ternary complex eIF-2GTP
Met-tRNA
with the 40 S
ribosomal subunit(13) . Based on these findings and unpublished
data, (
)Moldave and McLaughlin (14) suggested that
the PRT1 gene may encode a subunit of eIF-3. The cloning of
the PRT1 gene (15, 16) and the development of
a cell-free system in which Prt1 activity can be measured (17) allowed attempts to purify Prt1. Here, we show that Prt1
is a subunit of a protein complex that may be the yeast homologue of
mammalian eIF-3.
The optical density profile of the sucrose gradient showed a major peak of fast sedimenting material in fractions 7-9 followed by multiple peaks of more slowly migrating components (Fig. 1A). Individual fractions of the sucrose gradient were analyzed by SDS-PAGE and Western blotting. The Coomassie Brilliant Blue-stained gel revealed the copurification of five major polypeptides in fractions 7-9 (Fig. 1B, lanes3-6). They are most likely associated in a protein complex (see below). Two less abundant polypeptides (arrowhead and arrow in Fig. 1B) are probably not true subunits of the complex; the 50-kDa polypeptide (arrow) does not comigrate with the other polypeptides under the high salt concentration condition of the gradient but appears to migrate slower. The 116-kDa subunit (arrowhead) appears as a minor band after sucrose gradient centrifugation in this preparation (and not at all in another preparation, result not shown) but becomes more prominent upon further purification (compare Fig. 2B, lanes 2-4). This indicates that it may be a proteolytic digestion product of the largest polypeptide. The main polypeptides have apparent molecular masses of about 130, 80, 75, 40, and 32 kDa. The 75-kDa polypeptide is the Prt1 protein as judged from its reaction with the polyclonal rat anti-Prt1 antibody (Fig. 1C). The vast majority of Prt1 protein comigrates with the complex, and we estimate the protein complex to be at least 70% pure after sucrose density gradient centrifugation (Fig. 1B).
Figure 1: SDS-PAGE and Western blot analysis of sucrose density gradient fractions. A, optical density profile of sucrose gradient loaded with approximately 1.5 mg of protein of 300 mM KCl eluate fraction from phosphocellulose column (see ``Materials and Methods''). Sedimentation was from right to left. B, Coomassie Brilliant Blue-stained SDS-polyacrylamide gel. Lane1, marker proteins (prestained SDS-PAGE standards from Bio-Rad): phosphorylase b (106 kDa), bovine serum albumin (80 kDa), ovalbumin (49 kDa), carbonic anhydrase (32 kDa), soybean trypsin inhibitor (27 kDa), and lysozyme (17 kDa); lane2, 12 µg of 300 mM KCl eluate fraction from the phosphocellulose column; lanes 3-6, 20 µl of sucrose gradient fractions 6-9. C, peroxidase-stained Western blot. Samples were the same as in panelB.
Figure 2: Non-denaturing gel electrophoresis. A, lane1, marker proteins (see legend to Fig. 1); lane2, about 15 µg of sucrose density gradient-purified protein was fractionated on a polyacrylamide gel as described for SDS-PAGE, except that the gel, running buffer, and sample buffer did not contain SDS (non-denaturing condition). The gel was stained with Coomassie Blue. B, lane1, marker proteins; lane2, about 1.5 µg of protein complex after sucrose density gradient centrifugation; lane3, the slowest migrating band in panelA (arrow) was cut out, the gel piece loaded onto a SDS-polyacrylamide gel, and proteins fractionated by SDS-PAGE and stained with Coomassie Brilliant Blue; lane4, as lane3, but the gel was stained with silver.
To further substantiate that the Prt1 protein is associated with additional polypeptides in a stable protein complex we subjected the sucrose gradient-purified protein to non-denaturing gel electrophoresis (Fig. 2). Two main bands (arrow and arrowhead) and several minor bands were observed under non-denaturing conditions (Fig. 2A, lane2). We cut the main bands individually out of the gel and analyzed them by SDS-PAGE and staining with Coomassie Brilliant Blue (Fig. 2B, lane3) and with silver (Fig. 2B, lane4). Both produced the same band pattern; we obtained the five main bands discussed in Fig. 1B. Protein blotting verified that the 75-kDa band in the complex after non-denaturing gel electrophoresis is Prt1 protein (not shown). These findings support our observation that the 130-, 80-, 40-, and 32-kDa polypeptides form a stable protein complex with Prt1 protein.
Figure 3:
In
vitro translation. Extract of strain P501-1 was treated with
micrococcal nuclease and either kept at 0 °C (A) or
incubated at 37 °C for 5 min (B) before the addition of
[S]methionine (4 µCi/reaction). Where
indicated, 5 µg of total yeast RNA was added as mRNA. Incubation
mixtures (12 µl of total volume) were incubated at 25 °C, and
4-µl aliquots were assayed for methionine incorporation into
protein. A, lysate kept at 0 °C.
, minus mRNA;
, plus mRNA;
, plus mRNA plus 100 ng (0.28 pmol) of protein
complex;
, plus mRNA plus 200 ng (0.56 pmol) of protein complex. B, lysate preincubated at 37 °C.
, minus mRNA;
, plus mRNA;
, plus mRNA plus 100 ng (0.28 pmol) of protein
complex;
, plus mRNA plus 200 ng (0.56 pmol) of protein
complex.
Since it was shown
earlier that the yeast mutant strain prt1-1 is deficient
in ternary complex eIF-2GTP
Met-tRNA
binding to
the 40 S ribosomal subunit(13) , it was pertinent to test
whether Prt1-containing protein complex is able to stimulate this
reaction. We incubated preheated P501-1 extracts under
translation conditions for 5 min and fractionated them on sucrose
density gradients. A dose-dependent stimulation of methionine binding
to ribosomes was obtained (Fig. 4). Since the extract was
analyzed under translation conditions the subunit-joining reaction
converted most of the 40 S
Met-tRNA
complexes into 80
S initiation complexes.
Figure 4:
Methionine binding to ribosomes. Extract
of strain P501-1 was treated as described for Fig. 3,
heated for 5 min at 37 °C, and supplied with
[S]methionine (5 µCi/reaction). Incubation
mixtures (12 µl, about 5 pmol of ribosomes) were incubated at 25
°C for 5 min, diluted with 38 µl of dilution buffer (20 mM Tris-HCl, 100 mM KCl, 2 mM MgCl
),
and layered onto 4 ml of 5-30% sucrose density gradients in
dilution buffer. Gradients were centrifuged at 370,000
g for 90 min at 4 °C, and fractions (170 µl) were collected
and counted in a scintillation counter.
, no factor added;
, plus 100 ng (0.28 pmol) of protein complex;
, plus 200 ng
(0.56 pmol) of protein complex. Sedimentation was from right to left. The positions of the 40 and 80 S ribosomes in
the gradient are indicated.
These results show that Prt1 protein in the complex is active as a translation factor in vitro.
Figure 5: Growth of yeast transformants at 37 °C. Strain T92A was transformed with either the vector K10 (clone K10) or K10 containing the PRT1 open reading frame starting at position +490 in the sequence of Hanic-Joyce et al.(16) (clones 1-3). Transformants were plated on a YPGal plate (1% yeast extract, 2% peptone, 2% galactose), and the plate was incubated for 2 days at 37 °C.
The polypeptide Prt1 is most likely expressed from the AUG at position +490 in the sequence published by Hanic-Joyce et al.(16) and has therefore a molecular mass of 82.7 kDa. This is supported by (a) the S1 nuclease digestion experiments of Keierleber et al.(15) , (b) the finding that the polypeptide starting with methionine encoded by the AUG at position +490 is active in vivo (Fig. 5), and (c) the apparent molecular weight of Prt1 protein on SDS gels ( Fig. 1and Fig. 2). Prt1 is a subunit of a protein complex, which is stable under conditions of high salt concentration on sucrose density gradients (Fig. 1) and during non-denaturing acrylamide gel electrophoresis (Fig. 2). The abundance of the Prt1-containing protein complex in yeast cells was estimated to be about 0.2 copies/ribosome. This estimation is based on our recovery of protein complex during purification and the determination of the ribosome concentration by absorption at 260 nm after the high salt washing step. Since Prt1 protein is stably integrated in the protein complex and active in the restoration of translation in an extract in which endogenous Prt1 has been inactivated, we assume that Prt1 protein is active as a subunit of the protein complex.
Our data support (but do not prove) the earlier claim (14) that Prt1 is a subunit of the yeast homologue of mammalian eIF-3: (a) the purification scheme for Prt1 described in this work is very similar to the one used earlier by Trachsel et al.(24) to purify the mammalian factor; (b) the protein complex is a translation factor as shown previously (12, 13, 14) and in Fig. 3and Fig. 4of this work; and (c) like eIF-3, the Prt1-containing protein complex has RNA binding activity. The latter was shown by incubating the Prt1-containing protein complex with capped and uncapped (in vitro transcribed) RNA of 227 nucleotides in length containing an AUG translation initiation codon and measuring RNA-protein complex formation by filtration through a nitrocellulose filter. In this assay the Prt1-containing protein complex retains RNA on the filter (results not shown).
The Prt1-containing protein complex has fewer subunits and a lower molecular weight than mammalian eIF-3. The estimated molecular mass (340 kDa) is in agreement with data from Cigan et al.(25) showing that Prt1 is in a complex, which has a similar sedimentation behavior in glycerol gradients like yeast eIF-2B whose molecular mass is estimated to be 300 kDa(26) . We cannot exclude the possibility that the Prt1-containing protein complex contains additional subunits and that we lose these proteins during purification. Such proteins could reassociate with our purified complex in the in vitro translation system and restore active factor.
Based on our findings we believe that the Prt1-containing protein complex very likely represents the yeast homologue of mammalian eIF-3. The isolation of eIF-3 from the yeast S. cerevisiae is an important step toward the elucidation of the functions of this complex factor in translation initiation in eukaryotes.