(Received for publication, May 23, 1995; and in revised form, August 30, 1995)
From the
Yeast ribosomal protein L1 binds to 5 S rRNA and can be released from 60 S ribosomal subunits as an intact ribonucleoprotein particle. To identify residues important for binding of Saccharomyces cerevisiae rpL1 to 5 S rRNA and assembly into functional ribosomes, we have isolated mutant alleles of the yeast RPL1 gene by site-directed and random mutagenesis. The rpl1 mutants were assayed for association of rpL1 with 5 S rRNA in vivo and in vitro and assembly of rpL1 into functional 60 S ribosomal subunits. Consistent with previous data implicating the importance of the carboxyl-terminal 47 amino acids of rpL1 for binding to 5 S rRNA in vitro, we find that deletion of the carboxyl-terminal 8, 25, or 44 amino acids of rpL1 confers lethality in vivo. Missense mutations elsewhere in rpL1 also affect its function, indicating that multiple regions of rpL1 are important for its association with 5 S rRNA and assembly into ribosomes.
A useful approach to study ribosome biogenesis is to focus on a smaller, more compact particle within the ribosome and understand its assembly and function. This provides an opportunity to dissect the complex interactions that occur within the ribosome. One such particle that is a fundamental constituent of both prokaryotic and eukaryotic large ribosomal subunits is the complex between 5 S rRNA and ribosomal protein(s) that bind to it (Blobel, 1971; Chen-Schneisser et al., 1977; Smith et al., 1978; Nazar et al., 1979).
In the yeast Saccharomyces cerevisiae, this 5 S
ribonucleoprotein complex (5 S RNP) ()consists of ribosomal
protein L1 (rpL1 or L1, also known as L1a and YL3) and 5 S rRNA
(Mazelis et al., 1973; Nazar et al., 1979).
Similarly, in Xenopus, rat, chicken, and rice, the 5 S RNP
complexes are comprised of rpL5, the homolog of yeast rpL1, and 5 S
rRNA (Chan et al., 1987; Wormington, 1989; Kenmochi et
al., 1992; Kim and Wu, 1993). These eukaryotic 5 S rRNA-binding
ribosomal proteins are greater than 80% similar in sequence (Tang and
Nazar, 1991). The 5 S RNP complexes present in ribosomes of the
archaebacterium Halobacterium cutirubrum contain two proteins,
HL13 and HL19 (Smith et al., 1978), whereas those in Escherichia coli contain three proteins, EL5, EL18, and EL25
(Chen-Schneisser and Garrett, 1977). These bacterial proteins share
fewer sequence similarities with the eukaryotic 5 S rRNA-binding
ribosomal proteins.
5 S rRNA is the only rRNA known to form a stable RNP prior to its assembly into ribosomal subunits. In mammalian cells and Xenopus oocytes, 5 S rRNA interacts with rpL5 before assembling into ribosomes (Steitz et al., 1988; Guddat et al., 1990; Allison et al., 1991, 1993). Similarly, binding of 5 S rRNA to the three ribosomal proteins EL5, EL18, and EL25 is an obligatory step for its assembly into E. coli ribosomes in vitro (Yu and Wittmann, 1973).
Chemical and enzymatic
analysis of the 5 S RNP complexes, and binding studies with mutant 5 S
rRNA molecules have provided information about 5 S rRNA structure and
about sequences within 5 S rRNA that interact with ribosomal proteins
(Nazar, 1979; Garrett et al., 1981; Christiansen and Garrett,
1986; Yeh et al., 1988; Yeh and Lee, 1988; Guddat et
al., 1990; Allison et al., 1993). In contrast, very
little is known about sequences in the ribosomal proteins that interact
with 5 S rRNA. Chemical modification of lysines or arginines in yeast
rpL1 abolishes its ability to form a stable complex with 5 S rRNA in vitro, suggesting that the basic residues of rpL1 are
important for interaction with 5 S rRNA (Vioque et al., 1987).
Additional studies point to the importance of the rpL1 carboxyl
terminus in binding to 5 S rRNA. A CNBr-cleaved peptide fragment
containing the carboxyl-terminal 47 amino acids of rpL1 forms a stable
complex with yeast 5 S rRNA in vitro (Nazar et al.,
1979; Yaguchi et al., 1984). However, this binding is
nonspecific; this fragment could also bind 5.8 S rRNA and tRNAs in
vitro (Nazar et al., 1979). This 47-amino acid
carboxyl-terminal peptide fragment is rich in basic amino acids,
containing nine lysine and three arginine residues. Mutations in rpL1
that contained single methionine substitutions of either Lys-276,
Lys-279, or Lys-289 did not affect its ability to bind to 5 S rRNA in vitro; however, multiple substitutions of Lys-289 in
combination with other basic residues, particularly Arg-282 and
Arg-285, result in the formation of a nonfunctional rpL1 protein that
is deficient in 5 S rRNA binding in vitro (Yeh and Lee,
1995b). Because these residues are predicted to be located on the same
side of an -helix, it was proposed that 5 S rRNA may interact with
multiple contact sites located on one side of such a helical structure
in the carboxyl terminus of rpL1 (Yeh and Lee, 1995b).
In this
paper, we describe further experiments to functionally dissect rpL1 and
identify sequences that are important for its binding to 5 S rRNA or
assembly into ribosomes. Several lethal and temperature-sensitive
(Ts) alleles of the RPL1 gene encoding rpL1
were constructed by both site-directed and random mutagenesis. To study
how the mutations affect rpL1 function, we assayed the ability of the
rpL1 mutant proteins to assemble into ribosomes in vivo or to
bind to 5 S rRNA in vivo and in vitro. Sequences in
both the amino and the carboxyl terminus of rpL1 are required for its
interaction with 5 S rRNA. An intact carboxyl terminus of rpL1 is
necessary for its stable assembly into functional ribosomes.
Strain JWY3739 containing the
chromosomal rpl1-1::TRP1 null allele and plasmid
pRS315-RPL1-HA was transformed with pGAL1-RPL1 (Deshmukh et al., 1993); JWY3741 was derived from the
transformants upon loss of plasmid pRS315-RPL1-HA. JWY3755,
JWY3756, JWY3754, JWY3757, JWY3764, JWY3743, JWY3762, JWY3763, JWY3767
and JWY3753 were generated by transforming JWY3741 with mutagenized rpl1-HA plasmids as indicated in Table 1. Standard media
and techniques for growing yeast and bacteria were described by Sherman et al. (1986) and Moritz et al. (1990).
To clone the mutagenized fragments into an unmutagenized RPL1-HA gene, plasmid JWB2671 (LEU2) was digested with NcoI (site introduced at nucleotide 836) and PstI (at nucleotide 1056) to create a 220-base pair gap. This gapped plasmid was gel-purified and cotransformed with the gel-purified 380-base pair PCR amplified fragments into yeast strain JWY3740. Yeast transformants were plated on complete medium lacking leucine to select for in vivo recombination and repair of JWB2671 gapped plasmid with the mutagenized fragment. The plasmids containing mutant RPL1-HA genes were assayed for mutant phenotype by loss of the helper plasmid as described above.
We targeted mutagenesis initially to the 3` end of RPL1-HA, because a fragment corresponding to the
carboxyl-terminal 47 amino acids of rpL1 was previously shown to be
capable of binding to 5 S rRNA in vitro (Yaguchi et
al., 1984). We constructed four 3`-terminal deletion mutant
alleles, rpl1-HA-6, rpl1-HA-7, rpl1-HA-8, and rpl1-HA-9, by introducing a stop codon (Fig. 1). The rpl1-HA-6, rpl1-HA-7, and rpl1-HA-8 alleles, which
result in deletion of the carboxyl-terminal 44, 25, and 8 amino acids
of rpL1-HA, respectively, confer lethality in vivo (Fig. 1). However, the rpl1-HA-9 allele, which
contains a deletion of the carboxyl-terminal 2 amino acids of rpL1-HA,
does not result in lethality. These results are consistent with the
previous in vitro 5 S rRNA binding data (Yaguchi et
al., 1984) and indicate that the carboxyl-terminal 44 amino acids
are functionally important in vivo as well as in
vitro. An internal deletion of six amino acids, rpl1-HA-10 (103-108), which removes a domain (LLIARR), that is
conserved among the eukaryotic 5 S rRNA-binding ribosomal proteins also
causes lethality in vivo (Fig. 1).
Figure 1:
rpl1-HA mutant alleles. The
Tsrpl1-HA-1 to rpl1-HA-5 mutant
alleles contain single point mutations, as indicated. The rpL1-HA-6 to rpL1-HA-9 deletion mutant genes are truncated near the
3` ends of their coding regions at the indicated amino acids. The rpl1-HA-10 gene has an internal deletion of codons
103-108. The phenotypes conferred by these alleles in vivo were determined after plasmid
shuffling.
Since the
carboxyl-terminal deletions were lethal in vivo, we sought to
identify functionally important residues in that region. Random
mutations in the 3` end of RPL1-HA were generated by PCR
mutagenesis (Ma et al., 1987; Zhou et al., 1991;
Muhlrad et al., 1992). One Ts allele, rpl1-HA-5, was isolated; this allele contains a lysine to
glutamic acid change at codon 289 (Fig. 1).
To identify
additional rpl1-HA Ts alleles, we
mutagenized the RPL1-HA gene by forced nucleotide
misincorporation (Liao and Wise, 1990). Four rpl1-HA Ts
alleles (rpl1-HA-1, rpl1-HA-2, rpl1-HA-3, and rpl1-HA-4) were isolated, all of which contain a
single mutation (Fig. 1). The growth of the rpl1-HA Ts
mutants was compared by spotting the cultures
on plates incubated at different temperatures. The rpl1-HA-4 (G91R) and rpl1-HA-3 (V53G) alleles grew slower than the rpl1-HA-1 (K27E), rpl1-HA-2 (T28A), and rpl1-HA-5 (K289E) alleles at 37 °C (Fig. 2). The rpl1-HA-3 (V53G) allele was also cold-sensitive for growth at 13 °C (Fig. 2).
Figure 2: Temperature-sensitive and cold-sensitive alleles of RPL1. Equal numbers of cells from wild-type and mutant rpl1 cultures were spotted at low density onto three YEPD plates and incubated at 30, 37, and 13 °C. The plates grown at 30 and 37 °C were photographed after 2 days; the plate grown at 13 °C was photographed after 5 days. Yeast strains are: JWY3739 (RPL1-HA), JWY3750 (rpl1-HA-1), JWY3751 (rpl1-HA-2), JWY3749 (rpl1-HA-3), JWY3752 (rpl1-HA-4), and JWY3761 (rpl1-HA-5).
Figure 3: The temperature-sensitive rpl1-HA mutants contain diminished amounts of 60 S ribosomal subunits and accumulate half-mer polyribosomes at 37 °C. Polyribosome profiles are shown for JWY3739 (RPL1-HA), JWY3750 (rpl1-HA-1), JWY3751 (rpl1-HA-2), JWY3749 (rpl1-HA-3), JWY3752 (rpl1-HA-4), and JWY3761 (rpl1-HA-5) cells grown in rich medium at 23 °C and shifted to 37 °C for 2 h. Free ribosomal subunits and polyribosomes in cell extracts were separated on 7-47% sucrose gradients. Peaks representing 40 and 60 S ribosomal subunits and 80 S monoribosomes are labeled. Peaks representing two to six polyribosomes are also labeled for the wild-type profile. Vertical arrows indicate presumptive half-mer polyribosomes.
In certain rpl1-HA mutant strains, 60
S ribosome subunit assembly was disrupted even in the presence of
wild-type rpL1-HA protein. Yeast cells that express wild-type RPL1 from the P-RPL1 plasmid and mutant rpl1-HA alleles from another plasmid were grown in
galactose-containing medium, allowing expression of wild-type RPL1, under conditions that are nonpermissive for the rpl1-HA mutant alleles: 37 °C for the Ts
rpl1-HA alleles and 30 °C for the unconditionally
lethal rpl1-HA alleles. The polyribosome profiles of cells
expressing both rpl1-HA-1, rpl1-HA-2, rpl1-HA-4, and the wild-type RPL1 exhibited a
reduction in the amounts of 60 S ribosomal subunits compared with the
profile of wild-type cells (Fig. 4, compare A and D, E, or F). These dominant mutant phenotypes
indicate that the rpL1-HA-1, rpL1-HA-2, and rpL1-HA-4 mutant proteins
interfere with the assembly of wild-type 60 S ribosomal subunits, most
likely by competing with the wild-type rpL1. Differences in the size of
the 80 S monoribosome peak in these mutants are extract rather than
strain specific (data not shown). The 3`-terminal deletion allele
rpL1-HA-6 and the internal deletion allele rpL1-HA-10 do not interfere
with the assembly of wild-type 60 S ribosomal subunits (Fig. 4,
compare A with B and C). The other rpL1-HA
mutant proteins were not tested.
Figure 4:
The rpL1-HA-1, rpL1-HA-2, and rpL1-HA-4
mutant proteins disrupt the assembly of 60 S ribosomal subunits in the
presence of wild-type rpL1. Polyribosome profiles are shown for JWY3742 (RPL1-HA), JWY3743 (rpl1-HA-6), JWY3753 (rpl1-HA-10), JWY3755 (rpl1-HA-1), JWY3756 (rpl1-HA-2), and JWY3757 (rpl1-HA-4) cells grown in
galactose-containing medium. These cells express both the wild-type RPL1 and mutant rpl1-HA alleles under such
conditions. The JWY3755, JWY3756, and JWY3757 cells were grown at 37
°C to maintain the rpL1-HA Ts proteins at their
nonpermissive temperature. Peaks are labeled as in Fig. 3.
Using these yeast strains, that
express both the wild-type and mutant rpL1, we also examined the
polysome phenotype of cells expressing only the nonconditionally lethal
rpL1-HA alleles. We found that these rpl1-HA lethal mutants contain
fewer 60 S ribosomal subunits and accumulate half-mer polyribosomes
compared with the wild-type polyribosome profile, a phenotype that was
similar to that obtained for the rpL1-HA Ts mutants
(data not shown, Fig. 5, C and D).
Figure 5: Most of the different mutant rpL1-HA proteins can assemble into ribosomes. Shown are polyribosome profiles of JWY3742 (RPL1-HA), JWY3754 (rpl1-HA-3), JWY3753 (rpl1-HA-10), and JWY3762 (rpl1-HA-7) cells grown in galactose-containing medium and shifted for 2 h to glucose-containing medium. Cell extracts were separated on a 7-47% sucrose gradient, and fractions across the gradient were collected. rpL16 and HA-tagged mutant rpL1 were detected in these fractions by trichloroacetic acid precipitation of total protein and immunoblot analysis with anti-rpL16 and anti-HA-epitope antibodies. Peaks are labeled as in Fig. 3. Immunoblot lanes shown are correlated with the fractions from the polyribosome gradient.
To enable us to assay the mutant
rpL1-HA proteins at their nonpermissive conditions in the absence of
newly synthesized wild-type rpL1, we constructed yeast strains
containing the rpl1-1::TRP1 null allele, a plasmid that
expresses wild-type RPL1 from the repressible GAL1 promoter (P
-RPL1), and a second plasmid
bearing one of the mutant rpl1-HA alleles. Because the mutant
alleles are the only HA-epitope-tagged alleles in these cells, anti-HA
antibodies can specifically detect and immunoprecipitate the mutant
rpL1-HA proteins. We grew these cells in galactose-containing medium
under conditions that are nonpermissive for the mutant rpl1-HA alleles, 30 °C for the lethal alleles and 37 °C for the
Ts
alleles; these cells remain alive because
wild-type RPL1 is being expressed from the GAL1 promoter. Prior to harvesting the cells, we shifted these cells
from galactose- to glucose-containing medium for 2 h to rapidly
eliminate the synthesis of wild-type rpL1 in these cells and reduce
competition between the wild-type rpL1 and mutant rpL1-HA proteins for
binding to 5 S rRNA or assembly into ribosomes. All the mutant rpL1-HA
proteins except rpL1-HA-6 (
254-297) were detected in these
extracts by immunoblot analysis (see Fig. 6), indicating that,
with the exception of rpL1-HA-6, these mutant proteins are relatively
stable.
Figure 6: Binding of the mutant rpL1-HA proteins to 5 S rRNA in vivo. Yeast strains JWY3742 (RPL1-HA), JWY3755 (rpl1-HA-1), JWY3756 (rpl1-HA-2), JWY3754 (rpl1-HA-3), JWY3757 (rpl1-HA-4), JWY3764 (rpl1-HA-5), JWY3743 (rpl1-HA-6), JWY3762 (rpl1-HA-7), JWY3763 (rpl1-HA-8), and JWY3753 (rpl1-HA-10) were grown in galactose-containing medium and shifted for 2 h to glucose-containing medium. Cell extracts were treated with anti-HA-epitope antibodies to immunoprecipitate the mutant rpL1-HA proteins. L1-HA IP, the immunoprecipitated rpL1-HA proteins were detected by immunoblot analysis with anti-L1 RNP antibodies. 5 S rRNA co-IP, coimmunoprecipitation of 5 S rRNA with the mutant rpL1-HA proteins. 5 S rRNA was detected by hybridization with oligonucleotide 5S-2. These data were quantitated by densitometry (average of two experiments) and are represented in Table 2. To normalize for differences in stability of different mutant rpL1-HA proteins, the amount of 5 S rRNA coimmunoprecipitated with each mutant rpL1-HA protein was divided by the amount of that mutant protein precipitated.
To assay whether the mutant rpL1-HA proteins could assemble
into ribosomes, yeast cells were grown as described above, and cell
extracts were fractionated to resolve ribosomes and ribosomal subunits.
Fractions across these gradients were examined by immunoblot analysis
with anti-HA epitope monoclonal antibodies, to determine whether the
mutant rpL1-HA protein was present in peaks corresponding to the 60 S
ribosomal subunits, 80 S monoribosomes, or polyribosomes. The
distribution of rpL16, another 60 S ribosomal subunit protein, serves
as an internal control. We found that all five Ts rpL1-HA proteins could assemble into ribosomes; these proteins
were detected in fractions containing 60 S ribosomal subunits, 80 S
monoribosomes, and polyribosomes ( Fig. 5and data not shown).
This result is consistent with the fact that rpL1 is a 60 S ribosomal
subunit protein and indicates that the Ts
rpL1-HA
proteins are able to assemble into stable ribosomal subunits and
polyribosomes.
The rpL1-HA-7 (273-297) mutant protein was
detected in fractions corresponding to the 60 S ribosomal subunit peak
and to a lesser extent in the peak corresponding to the 80 S
monoribosomes. However, no rpL1-HA-7 was detected in the peaks
corresponding to polyribosomes (Fig. 5D). This result
indicates that although the rpL1-HA-7 mutant protein is able to
assemble into 60 S ribosomal subunits and to some extent in the 80 S
monoribosomes, ribosomes containing rpL1-HA-7 are apparently unstable
or nonfunctional. The rpL1-HA-10 (
103-108) mutant protein
could also assemble into ribosomes; this protein was detected in
fractions corresponding to the 60 S ribosomal subunits, 80 S
monoribosomes, and polyribosomes (Fig. 5C). However,
because the signal was weak, the rpL1-HA-10 protein may not be
assembling efficiently or may be partially unstable.
For the in vivo analysis, the mutant proteins were immunoprecipitated with anti-HA-epitope monoclonal antibodies from yeast cell extracts and the amount of 5 S rRNA coimmunoprecipitated with the protein was assayed ( Fig. 6and Table 2). To compensate for differences in stability of different mutant rpL1-HA proteins and for differences in the recognition of the various 5 S-L1-HA mutant complexes by the anti-HA antibodies, the amount of 5 S rRNA coimmunoprecipitated with a particular rpL1-HA mutant protein was divided by the amount of that protein precipitated. The amount of 5 S rRNA coimmunoprecipitated with the wild-type rpL1-HA protein was calculated as the control; the numbers obtained for various rpl1-HA alleles are expressed as percent of wild-type. The rpL1-HA-7 mutant protein that contains a deletion of the carboxyl-terminal 25 amino acids could bind 5 S rRNA to 52% of the wild-type levels in vivo; a smaller deletion of 8 amino acids at the carboxyl terminus (rpL1-HA-8) decreases binding to 65% of wild-type levels. These results indicate that, although the carboxyl terminus of rpL1-HA is necessary for binding, it is not the only 5 S rRNA binding domain in rpL1-HA. rpL1-HA-10 lacking amino acids 103-108 binds 5 S rRNA to only 7% of the wild-type levels. 5 S rRNA binding of the rpL1-HA-6 mutant protein could not be determined, because this protein was not detected in the extracts and hence was inferred to be unstable in cells (Fig. 6).
Among the five rpl1-HA Ts mutant proteins, rpL1-HA-3 (V53G) is most defective in binding 5
S rRNA in vivo; binding to 5 S rRNA is reduced to only 17% of
the wild-type levels. Binding of the rpL1-HA-1 (K27E) mutant protein to
5 S rRNA is 65% of wild-type levels; that for the rpL1-HA-2 (T28A)
mutant protein is 76% of wild-type levels. 5 S rRNA binding is only
marginally reduced in the rpL1-HA-4 (G91R) and rpL1-HA-5 (K289E)
mutants; these proteins bind 82 and 85% of wild-type amounts,
respectively, in vivo. These results indicate that valine 53
and amino acids 103-108 of rpL1-HA are very important in binding
5 S rRNA, either directly by interacting with 5 S rRNA or indirectly by
maintaining the structure of rpL1-HA.
To extend our in vivo analysis and to assay 5 S rRNA binding by a different criterion, we assayed a subset of the rpL1-HA mutant proteins for their ability to bind 5 S rRNA in vitro. An in vitro system for the study of rpL1 and 5 S rRNA binding has been described recently (Yeh and Lee, 1995a). One advantage of the in vitro analysis is that it enables us to assay 5 S rRNA binding of the mutant rpl1-HA proteins independently of other factors, such as differences in stability, ribosome assembly, or competition with wild-type rpL1, which could affect 5 S rRNA binding in vivo. These rpL1-HA mutant proteins were expressed in a coupled transcription-translation system from rabbit reticulocytes in the presence of exogenous yeast 5 S rRNA and assayed for 5 S RNP formation on an 8% nondenaturing polyacrylamide gel (Fig. 7). The 5 S RNP formation for each mutant was normalized to the amount of that mutant protein synthesized and expressed as a percent of the binding observed for the wild-type rpL1-HA protein.
Figure 7:
Binding of rpL1 to 5 S rRNA in
vitro, assayed by electrophoresis of RNP complexes on a
SDS-containing polyacrylamide gel (A) and a nondenaturing
polyacrylamide gel (B). The RNP was formed in a coupled
transcription-translation system from rabbit reticulocyte in the
presence of [H]leucine. The extent of RNP
formation was analyzed by electrophoresis followed by autoradiography. Lane 1, wild-type rpL1-HA. Lanes 2-5, mutant
rpL1-HA proteins with the following mutations: rpL1-HA-1 (Lys-27
Glu), rpL1-HA-2 (Thr-28
Ala), rpL1-HA-3 (Val-53
Gly), and
rpL1-HA-4 (Gly-91
Arg), respectively. These results were
quantitated by densitometry and are represented in Table 2. The
amount of RNP formed in each case (B) was normalized to the
amount of mutant rpL1-HA protein synthesized (A).
Consistent with the marginal 5 S rRNA binding of the rpL1-HA-3 and rpL1-HA-10 mutant proteins in vivo, these two mutant proteins did not form a detectable 5 S RNP complex in vitro as well (Fig. 7, data not shown). The percent RNA binding calculated for the rpL1-HA-1, rpL1-HA-2, and rpL1-HA-4 mutant proteins in vitro was 40, 60, and 50% of wild-type, respectively (Table 2).
Temperature-sensitive and lethal mutations in ribosomal
protein L1 were isolated by in vitro mutagenesis of RPL1-HA. Four carboxyl-terminal truncations and one internal
deletion of rpL1-HA were constructed, and five conditional lethal
alleles of RPL1-HA were isolated; one of the
Ts alleles, rpL1-HA-3 (V53A), was also
cold-sensitive. As has been suggested before, it is not uncommon to
find cold-sensitive mutations in RNA binding proteins (Zavanelli et
al., 1994).
All five yeast rpL1 Ts proteins
isolated contained mutations in amino acid residues that are highly
conserved among all the known eukaryotic 5 S rRNA binding ribosomal
proteins (Fig. 8). Since eukaryotic 5 S rRNAs have similar
tertiary structure, it is likely that the mechanism of interaction
between 5 S rRNA and these ribosomal proteins is also conserved in
evolution. One prediction of this hypothesis would be that rpL5
proteins from different species containing similar mutations would also
be defective for function.
Figure 8:
Alignment of sequences of the 5 S
rRNA-binding ribosomal protein from yeast, Xenopus, chicken and rat.
Arrows indicate the amino acids that are mutated in the five yeast rpL1-HA Ts alleles that are described in
this study.
The rpL1-HA mutant proteins could be defective for function, because the mutations cause structural changes in the protein. We have used the secondary structure prediction program described by Rost and Chris (1993, 1994) to determine whether mutations of rpL1-HA are likely to affect its secondary structure. None of the rpL1-HA point mutations were predicted to disrupt its major structural elements (data not shown). Previous studies have indicated that rpL1 is phosphorylated in vivo; up to two residues are thought to be phosphorylated per mol of rpL1 (Zinker and Warner, 1976; Campos et al., 1990). Although the significance of this phosphorylation is not yet known, it is possible that the rpL1-HA-2 (T28A) mutant protein and the rpL1-HA-6 and rpL1-HA-7 proteins that contain the carboxyl-terminal deletion are defective because they alter potential phosphorylation sites and thus affect rpL1 function.
None of the rpL1-HA
Ts mutants appear to accumulate any precursor or
aberrantly formed ribosomal subunit particles detectable by sucrose
gradient analysis. This observation contrasts with results obtained for
some bacterial ribosomal mutants, which accumulate aberrant ribosomal
subunit particles (Nashimoto and Nomura, 1970; Marvaldi et
al., 1979; Pichon et al., 1979). It appears that in
eukaryotes, any aberrantly formed ribosome particles are degraded
rapidly in vivo. Thus far, only one yeast ribosome assembly
mutant that accumulates aberrantly formed ribosomal subunit particles
has been described (Bayliss and Ingraham, 1974). This
streptomycin-sensitive yeast mutant accumulates a 28 S RNP containing
the 18 S rRNA. However, this RNP is not a precursor to the 40 S
ribosomal subunits; the molecular nature of this mutation is not known
(Bayliss and Ingraham, 1974).
The rpL1-HA-7 mutant protein, which lacks the carboxyl-terminal 24 amino acids, is present in fractions corresponding to the 60 S ribosomal subunits. However, very little mutant protein is present in the fractions corresponding to the 80 S monoribosome and none is detected in the polyribosome fractions (Fig. 5D). Thus, an intact carboxyl terminus of rpL1-HA is required for the formation of stable 80 S monoribosomes. Our results cannot distinguish whether these sequences are important for the formation of 80 S monoribosomes or for maintaining their stability. Other assays such as pulse-chase analysis of the mutant rpL1-HA protein would be able to distinguish whether the mutant proteins are degraded because they cannot assemble into ribosomes, or whether they assemble at normal rates, and subsequently, the ribosomes containing them are degraded.
Several RNA-binding proteins have discreet domains, such as the RRM sequences, RGG box, or arginine motifs, which are necessary and sufficient to interact with RNA (Mattaj, 1993). Our results indicate that yeast rpL1 does not interact with 5 S rRNA via any discreet domains; both amino- and carboxyl-terminal sequences are required for efficient interaction. These results are consistent with studies from other RNA-binding ribosomal proteins. Mutagenesis of the E. coli S8 protein identified 39 different mutations that cause defects in binding to 16 S rRNA. However, these mutations are not localized to any specific regions within the protein (Wower et al., 1992). A deletion of either the amino-terminal 14 amino acids or the carboxyl-terminal 6 amino acids of E. coli S20 reduces its binding affinity for 16 S rRNA in vitro, indicating that the rRNA-binding domain in S20 is complex (Donly and Mackie, 1988). Structural elements necessary and sufficient for yeast rpL25 to bind to domain III of 25 S rRNA in vitro are contained between amino acids 62 and 126 (Rutgers et al., 1991; Kooi et al., 1994).
One model for the biogenesis of the yeast L1-5 S RNP is that rpL1 interacts with 5 S rRNA first and subsequently the L1-5 S RNP assembles into 60 S ribosomal subunits. This is known to occur in mammalian cells and Xenopus oocytes where rpL5, a homolog of yeast rpL1, forms a L5-5 S RNP prior to its assembly into 60 S ribosomal subunits (Steitz et al., 1988; Guddat et al., 1990; Allison et al., 1991, 1993). Is binding of yeast rpL1 to 5 S rRNA a prerequisite for the assembly of rpL1 into 60 S ribosomal subunits? Perhaps a conformational change that occurs upon binding of rpL1 to 5 S rRNA is necessary for the L1-5 S RNP to subsequently assemble into 60 S ribosomal subunits; formation of the yeast L1-5 S RNP is known to result in a conformational change of the RNA (Yeh et al., 1988). This hypothesis would predict that mutant rpL1 proteins that are unable to interact with 5 S rRNA would also be unable to assemble into ribosomes. Alternatively if different sequences in rpL1 mediate its interactions with 5 S rRNA and assembly into ribosomes, some fraction of rpL1 that is not associated with 5 S rRNA could also independently assemble into ribosomes. Our results cannot distinguish between these two possibilities. The rpL1-HA-10 mutant protein that is greatly defective in binding to 5 S rRNA (7% of wild type) also appears to be defective in assembling into ribosomes (Fig. 5C and 6). However, the rpL1-HA-3 mutant protein that is defective in binding to 5 S rRNA (17% of wild type) does not appear to have an obvious assembly defect (Fig. 5B and 6). One caveat of this interpretation is that our assembly assay lacks the quantitation necessary for comparison of 5 S rRNA binding capability with ribosome assembly of these mutant proteins. Nevertheless, it would be useful to determine whether the rpL1-HA-3 mutant protein that is assembled into ribosomes is associated with 5 S rRNA. Isolation of rpL1 mutants that are completely defective for binding to 5 S rRNA in vivo may ultimately be necessary to determine whether prior binding of rpL1 to 5 S rRNA is a requirement for the assembly of rpL1 into 60 S ribosomal subunits.