The yeast SIS1 protein, a DnaJ homolog, is required for the initiation of translation

Cell, Vol. 73, 1175-1186, June 16, 1993, Copyright 0 1993 by Cell Press

The Yeast SISI Protein, a DnaJ Homolog, Is Required for the Initiation of Translation Tao Zhong’t and Kim T. Arndt’ *Cold Spring Harbor Laboratory Cold Spring Harbor, New York 11724 tGraduate Program in Genetics State University of New York at Stony Brook Stony Brook, New York 11794

Summary The S. cereviaiae S/S7 gene is essential and encodes a heat shock protein with similarity to the bacterial DnaJ protein. At the nonpermissive temperature, temperature-sensitive aia7 strains rapidly accumulate 80s ribosomes and have decreased amounts of polysomes. Certain alterations in 80s ribosomal subunits can suppress the temperature-sensitive phenotype of aia7 strains and prevent the accumulation of 80s ribosomes and the loss of polysomes normally seen under conditions of reduced SlSl function. Analysis of sucrose gradients for SISl protein shows that a large fraction of SIS1 Is associated with 405 riboaomal subunits and the smaller polysomes. These and other results indicate that SISl is required for the normal initiation of translation. Because DnaJ has been shown to mediate thedissociatlon of several protein complexes, the requirement of SISl in the initiation of translation might be for mediating the dissociation of a specific protein complex of the translation machinery. Introduction The Escherichia coli dnaJ and dnaK (encodes an HSP70 homolog) genes encode heat shock proteins (reviewed by Lindquist and Craig, 1988) that are essential for growth at temperatures above 42% (Sell et al., 1990). At the nonpermissive temperature, strains containing mutations in either dnaJ or dnaK are inhibited for both RNA and DNA synthesis (Wada et al., 1982). As shown by I. phage and Pl phage in vitro DNA replication systems, DnaJ can cooperate with DnaK to mediate specific protein-protein dissociations, perhaps by providing a chaperone-like function (Zylicz et al., 1989; Liberek et al., 1990; Wickner et al., 1991). DnaJ may also function independently of DnaK (Ohki et al., 1987; Straus et al., 1988), although how this might happen is not currently known. The yeast Saccharomyces cerevisiae is known at present to have five proteins with similarity to DnaJ (SEC63, Zuotin, YDJlIMAS5, SCJl, and SISl). The SEC63 and Zuotin proteins contain regions that are similar only to the more highly conserved amino-terminal third of DnaJ (Sadler et al., 1989; Zhang et al., 1992), while YDJlIMAS5 and SCJl are similar to DnaJ over their entire lengths (Caplan and Douglas, 1991; Atencio and Yaffe, 1992; Blumberg and Silver, 1991). SEC63 and YDJl may cooperate with heat shock protein 70 (HSP70)-type proteins

for the transport of proteins across membranes (Caplan et al., 1992; Sanders et al., 1992). The SlSl protein has similarity to DnaJ over the aminoterminal and carboxyl-terminal thirds of the proteins (Luke et al., 1991). In contrast, the middle third of SlSl is not related to the middle third of DnaJ. The SlSl protein performs an essential function and, like DnaJ, is a heat shock protein (Luke et al., 1991). To gain insight into the cellular function of SISl, we isolated mutations that could suppress the temperature-sensitive phenotype of a sisl-85 strain. One of these mutations occurred in the gene encoding a yeast homolog of the rat L35 ribosomal protein. A second alteration of large ribosomal subunits is also able to suppress the temperature-sensitive phenotype of asisl85 strain. Because of these results, we investigated the role of SlSl in translation. We show that SISl is required for the normal initiation of translation. Results Mutations in the Gene Encoding a Putative 80s Riboaomal Protein Suppress the TemperatureSensitive Phenotype of a aisl-85 Strain Strains containing the sisl-85 mutation (which results in the absence of amino acid residues 255-276, out of 352 total, of the SISl protein) are temperature sensitive for growth (Luke et al., 1991). To gain insight into the function of SISl , we isolated spontaneous Ts+ revertants of a sisl85 strain (see Experimental Procedures). One of the suppressor mutations, which we term ~0.~7-7 (for suppressor of sisl), was pursued further. Genetic analysis showed that the sosl-7 suppressor mutation segregates as a single nuclear gene, is unlinked to SISl, and causes a growth defect in either SW-85 or wild-type SlSl genetic backgrounds. We isolated the wild-type SOS7 gene from a YCp50 yeast genomic library (Rose et al., 1967) by complementing the slow growth phenotype of a sosl-1 S/S1 strain (see Experimental Procedures). Deletion analysis of the yeast insert of one of these SOSI-containing plasmids showed that the sosl-l-complementing region is located between nucleotides -254 and +950 (Figure 1A). Furthermore, deletion of the DNA sequences between the two Hindlll sites (located at +505 and +760) resulted in the inability to complement a sosl-7 strain. Conceptual translation of the DNA sequence of the complementing fragment, assuming an intron from DNA sequences +4 to +494, predicts a 120 amino acid protein with high similarity (57% identity) to the rat L35 ribosomal protein (Suzuki et al., 199Oj (Figures 1A and 2A). No other open reading frame is present on the -254 to +950 SOSlcontaining DNA fragment. The predicted SOS7 intron OCcurs immediately after the AUG initiation codon and has very good consensus 5’ and 3’ splice sequences and a TACTAAC sequence (all underlined in Figure 1A) (Woolford, 1989). In addition, the promoter region of SOS7 has DNA sequences (-292 to -281, underlined in Figure 1A)

Cell 1176

Figure

1. DNA and Predicted

Amino Acid Sequences

of SOS1 and SOS2

The sequence for SO.91 is shown in (A) and the sequence sequences, and TACTAAC sequences are underlined.

for SOS2

in (8). The potential

that are similar to the consensus RAP1 -binding site (ACACCCATACAllT; Woolford, 1991). An intron immediately after the AUG initiation codon and a RAPl-binding site in the promoter are typical of yeast genes encoding ribosomal proteins (Woolford, 1991; Woolford and Warner, 1991). A chromosomal deletion of SOS7 was prepared that replaces DNA sequences corresponding to amino acids 7-97 (of 120 total) of SOS1 with the LEU2 gene (see Experimental Procedures). Strains containing this Asosl muta-

::::::

EQQREAVRQLYKGKKYQPKDLRAKKTRALRRALTKFEASQVTEKQRKKQIAFFQRKYAIKA :.: .: :::::: : ::: ::::: :: ::: : : :: .: ::::.::

SOS1

QTQKENLRKFYKGKKYKPLDLRPKK~~F~L~.KHEEKLKTKKWRKERL;PLRKYAVKA

rat

wild type

5’ and 3’ intron

Figure 2. SOSl/SOS2lsSimilarto Ribosomal Protein

SOS1 rat

B

sites,

consensus

splice

tion have a slow growth phenotype (Figure 28). Interestingly, like the original sosl-7 mutation, deletion of the SOS7 gene also suppresses the temperature-sensitive phenotype of a sisl-85 strain (see Figure 3A). During Southern analysis of the Asosl strain, we found that the SOS7 probe hybridizes very well at high stringency to a DNA region distinct from SOS7. We cloned this DNA by hybridization using a SOS7 probe (SOS7 sequences +506 to +778). Conceptual translation of this DNA sequence

A MAGVKAYELRTKSKEQLASQLVDLKKEIAELWQKLS---RPSLPKIKWRKSIACVLTVIN : ::. ::::::

RAPl-binding

L35

L35

theRat

L35

(A) Similarity of the predicted SOS1 protein (which is identical to the predicted SOS2 protein) to the rat L36 ribosomal protein (Suzuki et al., 1990). Double dots indicate identical amino acids and single dots indicate conserved amino acids. (B) Growth phenotypes of isogenic wild-type (AY925), Asosl::LEU2 (CY1746). and AsasP:: URA3 (CY2509) strains. The strains were streaked onto YEPD plates and grown for 2.5 days at 30%.

SISI and the Initiation 1177

of Translation

C

YEp24

III

m/YEp24

Figure 3. Phenotypic pa61 Strains

Analysis

of

sisl

and

(A) Deletion of SOS1 or Sf82 suppresses the temperature-sensitive phenotype of a sisl-85 strain. A sis-85 strain was crossed to either a 38’C Asosl::LEU2 strain (CY873 x CYl746), a Aspb2::LEU2 strain (CY873 x CY2196). or a AtpMa::URAS strain (CY732 x CY2107). (5) Deletion of either SDS7 or SP82 sup9obl-F364L presses the temperature-sensitive phenotype of a pabl-F364L strain. A pabl-F364L strain 24’C (CY2195) wascrossed toeither a Asosl::LfU2 strain (CY1746), a AspbP::LEU2 strain (CY2196) or a ArpMa::URAS strain (CY2514). For both (A) and (6). the diploids were sporulated and the tetrads were dissected. Progeny of the indicated genotype were patched onto a YEPD plate and grown for 1 day at 24%. This ocd I-101 master plate was replica printed onto YEPD plates that were then grown at 24% or 36% pobl-F364L for 2 days. For each cross, a minimum of 20 tetrads were dissected. The temperaturesensitive phenotype of a representative progeny for each cross is shown. For each cross, all progeny of a given genotype had the same Ts phenotype. To showthat adsosl strain can pobl-F364L grow in the complete absence of the PAS1 gene, strain CY2194 (Ape61 plus wild-type PAS1 gene on YCpSO) was crossed to a Asosl::LElJ2 strain (CY1746). The diploid was cured of the PABlNCp50 plasmid and then sporulated. In 17 tetrad.% we obtained 10 Asosl::LEU2 Apabl::H/S3 progeny (one is shown in [El) and 18 Asosl::LEU2 progeny. Similar crosses using a Ask7 strain with S/ST on YCp50 showed that no Asos7::LEU2 Asisl::H/S3 progeny or AspbP::LfU2 Asisl::H/S3 progeny are obtained. In addition, Asosl Ask1 and Aspb2 Ask1 strains with sisl-85 on a YCpLO plasmid are not viable in the absence of the plasmid. (C) High copy number S/S1 inhibits the growth rate of apabl strain. YEp24 and YEp24 plasmids containing the wild-type S/S1 gene were transformed into strains CY2195 (pebl-F364L), CY2519 &cdl-lOI), or CY2522 (prfl-1). Transformants were streaked onto synthetic complete medium-uracil plates and grown at 24% for 3 days (CY2519 and CY2522) or 5 days (CY2195).

predicts a protein identical to the predicted SOS1 protein (Figure 16). Weterm thisgeneSOS2. LikeSOS7, theSOS2 gene has a predicted intron (with very good consensus 5’ and 3’ splice sequences and a TACTAAC sequence, all underlined in Figure 1 B) immediately after the AUG initiation codon. In addition, the promoter region of SOS2 has a potential RAPl-binding site (Figure 1B). Unlike a Asosl strain, a Asos2 strain (which replaces SOS2 DNA sequences corresponding to amino acids 7-97 with the URA3 gene) has only a slight slow growth phenotype (Figure 28). This effect is probably due to the fact that the levels of SOS7 RNA in a Asos2 strain are 3- to 4-fold higher than the levels of SOS2 RNA in a Asosl strain (data not shown). In addition, unlike deletion of SOS7, deletion of SOS2does not suppress the temperature-sensitive phenotype of a sisl-85 strain (data not shown). The map positions of the SOS7 and SOS2 genes, which are located about 28 CM apart on the left arm of chromosome IV, indicate that a region of that arm may have duplicated (see Experimental Procedures). When a Asosl:: LEU2 strain is crossed to a Asos2::URA3 strain, no Leu+ Ura+(Asos7::LEU2Asos2::URA3)progenywereobtained. For each of the 8 tetratype tetrads (in 14 tetrads total), the expected Leu+ Ura’ progeny did not give rise to a visible

colony, and microscopic analysis indicated that these spores had failed to divide. Therefore, in S. cerevisiae, the function of the SOSllSOSP rat L35 homolog isessential. It is common in S. cerevisiae for ribosomal proteins to be encoded by duplicated genes (Woolford and Warner, 1991). We will show below that mutations in SOS7 or SOS2 alter ribosome function. The Temperature-Sensitive Phenotypes of sisl and pabl Strains Are Suppressed by the Same Alterations in 80s Ribosomal Subunits SaCh8 and Davis (1989) have previously shown that a temperature-sensitive mutation in PAB7, which encodes the essential yeast poly(A)-binding protein (PAB), is suppressed by a mutation in any 1 of 7 genes, termed SPB7 through SPB7. PAB is required for the initiation of translation and for poly(A) shortening. All seven of the spb mutations reduce the levelsof 80s ribosomal subunits as determined by sucrose gradient analysis. SPB2 encodes ribosomal protein L48 (Sachs and Davis, 1989) and SPB4 encodes a putative ATPdependent RNA helicase that is required for the normal maturation of 25s ribosomal RNA (a component of 80s ribosomal subunits; Sachs and Davis, 1990). Therefore, alterations in either the structure

Cdl 1178

or levels of 60s ribosomal subunits can suppress the temperature-sensitive phenotype of pabl strains. Because of these results, we examined whether other alterations in 60s ribosomal subunits (in addition tososl-7 or dsosl) could suppress the temperature-sensitive phenotype of a s&l-85 strain. We found that deletion of SPBP (which encodes L46), but not deletion of RPL4A (which encodes L4A; Arevalo and Warner, 1990) can suppress the temperature-sensitive phenotype of a sisl-85 strain (Figure 3A). Likewise, the temperature-sensitive phenotype of a pabl-f364L strain (Sachs and Davis, 1989) can be suppressed by either the Asosl or Aspb2 mutation, but not by the Arpl4a mutation (Figure 38). Therefore, the temperature-sensitive phenotypes of sisl-85 strains and pabl-F364L strains are suppressed by the same alterations in 60s ribosomal subunits. Sachs and Davis (1989) have previously shown that the Aspb2 mutation allows a strain to grow in the absenceof the PAB7 gene. The Asosl mutation also allows a strain to grow in the complete absence of PAB7 (Figure 38). In contrast, neither the Asosl nor the AspbP mutation allows a strain to grow in the absence of the S/S7 gene (Figure 38). Since PAB is required for the initiation of translation, we tested whether a temperature-sensitive mutation in genes encoding other translation initiation factors could be suppressed by a Asosl mutation. These experiments showed that the Asosl mutation is not able to suppress the temperature-sensitive phenotype of a gcdl-707, gcdl-507, ptttl-7, orprtl-63 strain (data not shown). GCD7 encodes a probable component of eukaryotic initiation factor (elP)-PB (Cigan et al., 1991), and PM7 encodes a probable component of elF-3 (Keierleber et al., 1986). Therefore, the ability of an alteration in 60s ribosomal subunits to suppress defects in translation initiation is somewhat specific to sisl-85 (see below) and pabl mutations. Additional evidence linking S/S7 and PAB7 to a common process is that under permissive growth conditions(24%), high copy number S/S7 inhibits the growth rate of apablF364L strain. In contrast, high copy number SlS7 does not inhibit the growth rate of wild-type, gcdl-707, gcdl-507, prtl-7, or prtl-63 strains (Figure 3C and data not shown). This genetic interaction between high copy number SlS7 and thepab7+364L mutation suggests that SISl and PAB might function in the same pathway or in interacting pathways. SlSl Is Required for the Normal Initiation of Translation The above results caused us to examine whether SlSl, like PAB, is required for the initiation of translation. When shifted to 38%, sisl-85 cells incorporated into acidprecipitable peptides 50% of the amount of [%]methionine that isogenic wild-type S/S7 cells incorporated (data not shown). To examine the translational defect in the sisl85 cells more closely, we prepared extracts from isogenic sisl-85 and wild-type SlS7 cultures and fractionated the extracts on sucrose gradients. At the permissive temperature (24%), a sisl-85 strain had a relatively normal ribosome and polysome profile, except that the levels of polysomes (Figure 4, the peaks to the right of the 80s ribosome

peak) were slightly lower than those from an isogenic wildtype S/S7 strain. After 30 min at 37.3%, a sisl-85 strain accumulated high levels of 80s ribosomes and had reduced levels of polysomes in comparison with those of an isogenic wild-type S/S7 strain (Figure 4). Strains containing asisl-86 mutation (which deletes SISl amino acids 286-307, out of 352 total) were also temperature sensitive for growth (Luke et al., 1991). After 30 min at 37.3%, a sisl-86 strain accumulated high levels of 80s ribosomes and had reduced levels of polysomes, in comparison with an isogenic wild-type S/S7 strain (Figure 4). Therefore, two different temperature-sensitive mutations in S/S7 give rise to alterations in polysome profiles that are diagnostic for a defect in the initiation of translation. The requirement for SISl in translation is probably direct, since 80s ribosomes accumulated and polysomes levels decreased by 10 min after the sisl-85 culture was shifted to the nonpermissive temperature (Figure 5A). If elongation was blocked (by addition of cycloheximide) prior to shifting the sisl-85 cells to the nonpermissive tem-

CY733 s151-86

sedimentalon

Figure 4. sisl-85 Have Decreased perature

-

and sisl-88 Strains Accumulate 80s Ribosomes and Amounts of Polysomes at the Nonpermissive Tem-

Culturesofthree isogenicstrains,CY457(wild-typeSIS7) CY732(sisl85) and CY733 (sisl-86) were grown at 24% in YEPD medium to an OD, of 0.4. At this time, the cultures were shifted to a 37.3% water bath. Parallel cultures were maintained at 24%. After 30 min, cycloheximide was added (to 60 &ml final concentration), and the cells were harvested. Extracts were prepared and loaded onto sucrose gradients as described in Experimental Procedures,

SlSl and the Initiation 1179

of Translation

A Time course, 37°C

B CYH, then 37OC for 30min.

Figure 5. Analysis somes of a &l-85

:Y732 ,151-85 II

i

h

C. High

Salt;

sisl-8!$

of the Ribosomes Strain

and Poly-

(A)Time courseof 80s ribosome accumulation and polysome decrease when a sisl-85 strain is shifted to 37%. Four parallel cultures of strain CY732 were grown at 24% in YEPD medium to an ODaa of 0.4. At this time, three of the cultures were shifted to a 37% water bath. At 10, 20, or 30 min after the shift to 37%, cycloheximide was added and the cells were immediately harvested. Extracts were prepared and loaded onto sucrose gradients, The arrow labeled P indicates the first polysome for the 30 min sample. After 30 min at 37OC, the polysomes sedimented more slowly. The mechanism causing this effect, which is not seen for a gcdl-701 strain (data not shown), is not known. (B)Addition of cycloheximide (CYH) tothesisl85 culture prior to the temperature shift prevents the accumulation of 80s ribosomes and the decrease in polysomes. Cultures of strains CY457 (wild type) or CY732 (SW-85) were grown in YEPD medium to an Ot& of 0.4. At water bath. After 30 min at 37%, the cells were

this time, cycloheximide was added (to 60 mglml final) and the cultures were shifted to a 37% harvested. (C) The 60s ribosomes that accumulate in the absence of normal SlSl function are dissociated into 40s and 60s subunits by a high concentration of NaCI. Two parallel cultures of strain CY732 (sisl-85) were grown at 24% in YEPD medium. At an ODaa of 0.4, one of the cultures was shifted to a 37% water bath. After 30 min, cycloheximide was added to both cultures and the cells were harvested. The extracts were prepared with 0.6 M (final concentration) NaCl buffer, and sucrose gradients were prepared with 0.7 M NaCl buffer. For all three panels, extracts were prepared and loaded onto sucrose gradients as described in Experimental Procedures.

perature, the polysome levels did not decrease and 80s ribosomes did not accumulate (Figure 58). Therefore, when elongation was not blocked (as in Figures 4 and 5A), the 80s ribosomes that accumulated in the absence of normal SISl function were from ribosomes within polysomes that had terminated translation. Because initiation was blocked in the absence of normal SISl function, 80s ribosomes accumulated. The high levels of 80s ribosomes that accumulated in the absence of normal SlSl function could have been 80s couples (inactive80S ribosomes not bound to mRNA), 80s preinitiation complexes, and/or 80s monoribosomes (a monoribosome is an mRNA with a single 80s ribosome engaged in elongation). A high concentration of salt dissociates 80s couples and 80s preinitiation complexes into 40s and 80s subunits. In contrast, ribosomes that are in the process of elongation are not dissociated into 40s and 80s subunits by a high concentration of salt (Martin, 1973). When the si87-85 strain was grown at 24% most of the ribosomes were in the process of elongation. Preparation of the extract using a buffer containing a high concentration of salt generated only modest amounts of 40s and 80s subunits (Figure 5C). In contrast, when the sisl-85 strain was grown at 37OC for 30 min, elongation and termination could continue, but initiation was blocked. Under these conditions, the levels of salt-resistant polysomes decreased and high levels of 80s ribosomes accumulated (see Figure 4). When the extract was prepared using a buffer containing a high concentration of salt, almost all of the 80s ribosomes were dissociated into 40s and 60s subunits (Figure 5C). Therefore, the 80s ribosomes that accumulate in the absence of normal SlSl

function are either 80s preinitiation couples.

complexes

or 80s

A sosl Mutation Prevents the Accumulation of 80s Monorlbosomes and the Decrease of Polysomes in a sisl-85 Strain at 37% By itself, the Asosl mutation resulted in levelsof 60s ribosomal subunits, 80s ribosomes, and polysomes that were lower than those of an isogenic wild-type strain (Figure 6A). In addition, the Asosl strain had higher levels of 40s subunits than the wild-type strain (Figure 6A), presumably because of its lower level of 60s subunits. In the absence of sufficient 60s subunits, either extra 43s preinitiation complexes or extra 40s subunits accumulate on the mRNA, giving rise to halfmer polyribosomes (labeled H in Figure 8) (Rotenberg et al., 1988). In agreement with the faster growth rate of a Asos2 than a Asosl strain, a Asos2 strain had less severe alterations of the ribosomes and polysomes than a Asosl strain (Figure 8A). These alterations in ribosomes and polysomes by a Asosl or Asos2 mutation confirm that SOS7 and SOS2 encode 60s ribosomal subunit proteins. Since the Asosl mutation can suppress the temperature-sensitive phenotype of a sisl-85 strain, we determined whether the Asosl mutation alters the polysome profile of a sisl-85 strain. When grown at a permissive temperature (24%), a sisl-85 Asosl strain had slightly lower levels of polysomes than a S/S7 Asosl strain (Figure 6B). Therefore, in both As087 (Figure 8B) and wild-type SOS1 (see Figure 4) genetic backgrounds, the sisl-85 mutation resulted in slightly lower levels of polysomes at 24% (as compared with a S/S7 strain). Remarkably, when the

Cell 1160

Figure

6. Effect

of the dsosf

and dsos2

Mutations

(A) The effect of the deletion of SOS7 or SOS2 on ribosomes and polysomes in a wild-type S/S7 strain. Cultures of three isogenic strains, AYg26 (wild type), CY1741 (dsosl::LEU2), and CY2509 (dsos2:: URA3), were grown in YEPD medium at 30% to an ODW of about 0.4. Cycloheximide was added, and the cells were harvested. (E) Deletion of SOS7 prevents the accumulation of 60s ribosomes and the decrease in polysomes normally seen in the absence of normal SlSl function. Two isogenic strains, CY1741 (dsosl::LEU2) and CY2012 (sisl-85 dsosl::LEU2), were grown in YEPD medium to an ODaaof 0.4. At this time, thecultures were shifted to a 37% water bath. Parallel cultures were maintained at 24°C. After30 min, cycloheximide was added and the cells were harvested. For both panels, sucrose gradients were performed as described in Experimental Procedures. The arrows labeled H indicate halfmer poly ribosomes that accumulate when the levels or structure of 60s ribosomal subunits are altered.

sisl-85 Asosl strain was shifted to 37% 80s ribosomes did not accumulate, and the levels of polysomes did not decrease (Figure 8B). Therefore, alteration of either the levels or structure of 80s ribosomal subunits by the Asosl mutation suppresses the temperature-sensitive phenotype of a sisl-85 strain by eliminating the block in the initiation of translation that normally occurs in the absence of normal SISl function. Some of the SlSl Protein Is Associated with 40s Subunits and the Smaller Polysomes That SlSl is required for the initiation of translation raises the possibility that SIS1 might associate with ribosomes.

To examine this possibility, we sedimented yeast extracts on sucrose gradients. The gradients were dripped through an ultraviolet monitor, and fractions were collected and assayed for SISl protein by Western analysis. When an extract prepared from a wild-type strain was used, the SISl protein sedimented throughout much of the sucrose gradient, including positions corresponding to 40s subunits, 80s subunits, 80s ribosomes, and the smaller polysomes (Figure 7A). Therefore, under the buffer conditions used in this experiment (20 mM HEPES [pli 7.51, 10 mM KCI, 5 mM MgCI,), the SISl protein was present in high molecular weight complexes. The great majority of the other proteins present in the extract remained at the top of the gradient (fractions 1, 2, and 3) as seen by staining of the blot with Coomassie brilliant blue (data not shown). To shift the sedimentation rate of ribosomes present within the polysomes, we treated the extract with RNAase A, which digests the mRNA linking the ribosomes together in the polysomes. When an extract prepared from wildtype cells was treated with RNAase A prior to loading onto the sucrose gradient, the levels of 40s subunits were reduced, the peak of 80s subunits and polysomes were almost completely eliminated, and the levels of 80s ribosomes were greatly increased (Figure 78). These 80s ribosomes were composed of the original 80s ribosome population (assuming they are not destroyed by the RNAase A; seen in Figure 7A) and ribosomes derived from the RNAase A-digested polysomes. The broad rightward slope probably indicates that the polysomes were not completely digested into monosomes by the RNAase A. When the extract was treated with RNAase A, most of the SlSl protein sedimented in the 80s ribosomal fractions (Figure 78). Therefore, in the absence of RNAase treatment (Figure 7A), the SISl protein that cosediments with the polysomes (and possibly with the 80s ribosomal subunits) is present in high molecular weight complexes whose sedimentation rate is dependent on an intact RNA species. To determine whether the RNAase A-sensitive SISlcontaining complexes are ribosomal complexes, we used a second method for altering the sedimentation of polysomes. The PRT7 gene product is a probable component of elF-3 and is required for the initiation of translation (Keierleber et al., 1988). When a temperature-sensitive prfl-7 strain was shifted to 37% for 30 min, the polysomes were almost completely absent and 80s ribosomes accumulated to very high levels (Figure 7C); the levels of 405 and 80s subunits were slightly reduced. Under these conditions, the SISl protein no longer sedimented to positions that would normally correspond to polysomes (fractions 1O-l 7; Figure 7C). Two control experiments show that the absence of detectable SISl in fractions lo-17 was due to the absence of polysomes. First, when a wild-type strain was shifted to37% for 30 min, the ribosome and polysome distribution and the sedimentation of SlSl was essentially the same as in Figure 7A (data not shown). Therefore, the temperature shift itself does not cause an alteration in the sedimentation of SlSl. Second, if cycloheximide was added to the prtl-7 culture just prior to shifting to 37%, elongation was

SlSl and the Initiation 1181

of Translation

Figure 7. Cosedimentation of the SlSl with 405 Subunits and Polysomes

Wild type +RNose

Protein

(A and B) An extract was prepared from wildtype strain AY925 grown at 24% in YEPD medium and divided into two portions. To one portion of the extract (used in [B]), RNAase A was added to 300 ug/ml final concentration. To the other portion (used in [A]), the same volume of buffer was added. Then the extracts were incubated at 4% for 40 min and loaded onto sucrose gradients. (C) Strain CY2522 (pr?l-I) was grown at 24% to an OD, of 0.4 and shifted to a 37% water bath. After 30 min, cycloheximide was added and the cells were harvested. Extracts were prepared and loaded onto a sucrose gradient. (D) The extract was prepared from strain CY2952 (&x286). (E) The extract was prepared from strain CY1741 (dsosl). For this gradient, smaller fractions were collected (25 total fractions) to increase the resolution in the 50s region. Fractions 1, 2, and 21-25 are not shown. The vertical OD- scale of(E) is expanded 2.5fold from that of (A-D). For all panels, fractions were collected from the sucrose gradients and probed for SlSl protein as described in Experimental Procedures.

---

blocked so that the polysomes levels did not decrease and 80s ribosomes did not accumulate. Under this condition, SlSl sedimented in the polysome fractions, with distribution essentially identical to that shown in Figure 7A (data not shown). Therefore, lowering the levels of polysomes by two independent methods (RNAase A treatment in vitro or a block of translation initiation in vivo) results in a reduction of the amount of SlSl in the gradient fractions that would normally correspond to polysomes (compare Figures 76 and 7C with Figure 7A). These results suggest that SlSl is associated with the smaller polysomes. To determine whether the sedimentation of SlSl in the 40s subunit fractions is due to association of SlSl with the 40s subunits, we used a strain containing a deletion of RfS28B, 1 of 2 genes that encodes ribosomal protein S28 (a component of 40s subunits). When the extract was prepared from the Arps28b strain, the 40s subunit peak was almost completely absent (Figure 7D), and free 60s subunits accumulate to high levels, which caused the 60s peak to overlap with the 80s peak. That SlSl was almost completely absent in fractions 3, 4, and 5 (Figure 7D), which would normally correspond to the 40s subunit peak, suggests that SlSl associates with 40s subunits.

We also examined the interaction of SISl with 60s subunits. Reducing the 60s subunit peak as a result of the RNAase A treatment lowered the levels of SISl in the gradient fractions that would normally correspond to 60s subunits (Figure 7B). However, increasing the levels of free 60s subunits (owing to deletion of RPSPBB) did not result in a correspondingly large increase in the levels of SlSl in the 6OSsubunit fractions. (For Figure 7D, the larger 60s subunit peak, fraction 7, had less SISl than the smaller 80s peak, fraction 8.) Perhaps the amount of SISl that can associate with free 60s subunits is limiting. To investigate further the possible interaction of SlSl with 60s subunits, we fractionated extracts prepared from Asosl and Asos2 strains (which have reduced levels of 60s subunits). For the Asosl and Asos2 extracts, we consistently observed a decrease in the levels of SlSl in the 60s subunit fractions (compared with wild-type extracts; data not shown and Figure 7E, in which smaller fractions were collected from the gradient to increase the resolution). Although we were not able to obtain conditions that would completely eliminate the 60s subunit peak (to show that SISl would completely disappear from the fractions corresponding to 60s subunits), these results raise the

Cell 1182

possibility that some SE1 is associated units.

with the 60s sub-

Discussion SlSl Is Required for the Initiation of Translation Alterations in 60s ribosomal subunits suppress the temperature-sensitive phenotype of a SW-85 strain. This finding led us to investigate whether SlSl is required for translation. In the absence of normal SlSl function, the polysome levels decrease and 80s ribosomes accumulate to high levels. Because these 80s ribosomes are dissociated into 40s and 60s subunits by a high concentration of salt, they are most likely 80s preinitiation complexes and/or free 80s couples. 80s preinitiation complexes would accumulate if SISl functions at a step required for progression from an 80s preinitiation complex to elongation. Alternatively, blocking initiation at almost any step will increase the levels of free 80s couples. If elongation and termination continue under conditions at which initiation is blocked, 40s and 60s subunits increase in levels and join to form inactive 80s couples (see Hartwell and McLaughlin, 1969; Cigan et al., 1991; Foiani et al., 1991). The accumulation of 80s ribosomes and the decrease in polysome levels that occur in the absence of normal SISl function are prevented by blocking elongation with cycloheximide. Addition of cycloheximide also prevents the accumulation of 80s ribosomes and the decrease in polysome levels that occurs in the absence of GCDl, GCDP, or PRTl function (Hattwell and McLaughlin, 1969; Cigan et al., 1991; Foiani et al., 1991). Therefore, the decrease in polysome levels that takes place in the absence of normal SlSl function is most likely due to the continuation of the elongation of nascent polypeptide chains, followed by the eventual termination of translation. The ribosomes that dissociate from the polysomes accumulate to high levels in the absence of normal SISl function because the frequency of new cycles of initiation is greatly reduced(foreitherall mRNAsoralargesubsetof mRNAs). Therefore, at 37% the primary translational defect of sisl strains is in some step(s) required for the initiation of translation. However, we cannot exclude an additional role for SISl in elongation. At What Initiation Step Does SlSl Function? Alteration of the structure or levels of 60s ribosomal subunits by the Asosl mutation completely prevents the accumulation of 80s ribosomes and the decrease in polysome levels that normally occur in the absence of normal SISl function. Perhaps an excess of 40s subunits (due to lower levels of 60s subunits) overcomes the requirement for normal SISl function. If so, it is possible that SISl functions to dissociate 80s ribosomes into 40s and 60s subunits (especially considering that SISl is a DnaJ homolog). Free 40s subunits are required for the initiation process and are generated from 80s ribosomes via the action of elF-3 and additional factors. As an alternative to lower levels of 60s subunits, an altered structure of 60s ribosomal subunits might overcome the requirement for normal SlSl function. SlSl could be required at some step at which

60s subunits join with the 40S-mRNA complex to form the 80s preinitiation complex and progress into elongation. An altered structure of 60Ssubunits could reduce the possible requirement of SlSl in this process. In support of an altered structure of 60s subunits overcoming the requirement for normal SlSl function, the Aspb2 mutation is better than the Asosl mutation as a suppressor of the temperature-sensitive sisl-85 mutation (see Figure 3A), even though a Aspb2 strain has higher levels of 60s subunits than a Asosl strain (data not shown). Interestingly, mutations in PAB7 are suppressed by the same mutations in SOS7 and SPt32 that suppress the sisl85 mutation. In yeast, PAS is required for the normal initiation of translation (Sachs and Davis, 1989). Together, the PAB protein and the 3’ poly(A) sequence functionally act as an enhancer of translation initiation (Munroe and Jacobson, 1990) although the mechanism by which PAB stimulates initiation is currently unknown. Not only are mutations in S/S7 and PAB7 suppressed by the same alterations in 60s subunits, but overexpression of SlSl inhibits the growth rate of a pabl-F364L strain (but not that of wild-type, gcdl, oroft strains). These results suggest that SlSl and PAB either function at the same initiation step or function in different, but interacting, initiation steps. That the Asosl or Aspb2 mutation can allow viability in the complete absence of PAB7, but not in the complete absence of SlS7, leaves two possibilities. One is that SISl , unlike PAB, is required for some essential process in addition to the initiation of translation. The second possibility is that SlSl is essential only for translation, but that alterations in 60s ribosomal subunits cannot compensate for the complete absence of SlSl (unlike the complete absence of PAB). For either possibility, the SISl-85 protein would have to be slightly functional at the nonpermissive temperature. Association of SlSl with Ribosomes Analysis of extracts fractionated on sucrose gradients suggests that SISl is associated with 40s ribosomal subunits and the smaller polysomes. (It is also possible that a small amount of SlSl is associated with 60s ribosomal subunits.) The association of SISl with 40s subunits would be consistent with a direct role for SlSl in the translation initiation process. A fraction of SISl also cosediments with 80s ribosomes and may be associated with some form of 80s ribosome. Compared with the fraction of SlSl that cosediments with 80s ribosomes for a wild-type extract, a slightly larger fraction cosediments with the very high levels of 80s couples that accumulate in a prtl-7 strain at the nonpermissive temperature (compare Figures 7C and 7A). However, given the very high levels of free 80s couples that accumulate in the pal-7 strain, either the fraction of SISl that can associate with free 80s couples is limiting, or SlSl does not efficiently associate with free 80s couples. This line of reasoning would suggest that much of the SISl that cosediments with 80s ribosomes might be associated with 80s preinitiation complexes, which are formed when the 60s subunit joins with the 40s subunit-mRNA complex. Since SISl is most likely associated with 40s subunits, the presence of SlSl in 80s

SISl and the Initiation 1183

of Translation

preinitiation complexes would be expected, unless SlSl rapidly dissociates from the 40s subunit when the 60s subunit joins with the 40s subunit-mRNA complex. Also, in ten sucrose gradients, more SlSl is reproduciblyassociated with the smaller than the larger polysomes (Figure 7A and data not shown). This result might be due to the fact that smaller polysomes have agreater ratioof initiating ribosomes to total ribosomes. For each ODzyl unit of polysome, the smaller polysomes contain a greater amount of initiating ribosomes than the larger polysomes. One possible explanation is that SlSl is associated with 80s ribosomes that are in the process of initiation. After a ribosome has progressed into elongation, SlSl may dissociate. The cosedimentation of SISl with ribosomes is sensitive to the salt concentration of the gradient buffers. The sucrose gradients shown in Figure 7 used extract and gradient buffers containing 20 mM HEPES (pH 7.5), 10 mM KCI, 5 mM MgCl2. The addition of 0.3 M sorbitol and 10% glycerol to the extract and gradient buffers does not alter the sedimentation profile of the ribosomes or the SISl protein (data not shown). If the concentration of KCI in the extract buffer is increased to 50 mM (or even 100 mM), keeping the concentration of KCI in the gradient buffers at 10 mM, the sedimentation profile of both the ribosomes and the SISl protein is similar to that in Figure 7A (data not shown). In contrast, if the concentration of KCI in both the extract and gradient buffers is increased to 50 mM, the sedimentation profile of ribosomes is not altered, but most of the SlSl is found near the top of the sucrose gradients (mostly in fractions l-5) (data not shown). However, under these conditions, about 5% of the SlSl protein still cosediments with 80s ribosomes and the smaller polysomes. We interpret these results as follows: the SISl protein and the ribosomes in the extracts are present at relatively high concentrations (similar to in vivo conditions). In the extract, under equilibrium conditions, most of the SlSl is associated with various forms of ribosomes, even at KCI concentrations up to 100 mM. However, when the SISlribosomal complexes sediment through the sucrose gradient, any SlSl that dissociates from the ribosomes will remain near the top of the gradient. Low concentrations of KCI in the gradient buffers are necessary to maintain the association of SlSl with the ribosomes during the course of sedimentation (3.5 hr at high hydrostatic pressure). Because we do not know how the conditions of our in vitro experiments relate to conditions inside a yeast cell, it is difficult to state the physiological significance of the ionic strength-dependent association of SlSl with ribosomes. SlSl as a DnaJ Homolog For in vitro hand Pl phage DNA replication systems, DnaJ functions to mediate the dissociation of specific protein complexes (Zylicz et al., 1989; Liberek et al., 1990; Wickner et al., 1991). That SISl has similarity to DnaJ suggests that SISl might function for the initiation of translation by mediating the dissociation of a specific protein complex. There are many steps during the initiation process that might require such a function of SISl.

In many systems, DnaJ homologs function along with a DnaK/HSP70 homolog. In budding yeast, the HSP70 homologs SSBl and SSB2 have been shown to be important for normal translation elongation and to associate with polysomes (Nelson & al., 1992). That SlSl is essential for normal initiation while SSBl and SSBP are important for normal elongation would suggest that SlSl and the SSB proteins do not functionally interact. However, it remains to be determined whether SISl cooperates with SSBl and SSB2 (or any other HSP70 homolog) during the translation process. Could bacterial DnaJ function in translation? When a dnaJ mutant is shifted to the nonpermissive temperature, the stringent response is induced (Wada et al., 1982). The stringent response (which results in the accumulation of ppGpp, which binds to RNA polymerase and alters RNA synthesis) of dnaJ mutants at the nonpermissive temperature is prevented by there/A mutation (Itikawa et al., 1986). The RelA protein is associated with ribosomes and synthesizes ppGpp during conditions of inhibition of protein synthesis. Therefore, the fact that dnaJ mutants induce the stringent response at the nonpermissive temperature suggests that DnaJ might be required for normal protein synthesis in bacteria. The finding that a DnaJ homolog, SlSl, is required for the initiation of translation adds further evidence to the notion that heat shock proteins are required for fundamental cellular processes. The elucidation of the precise role of SlSl for the initiation of translation will require a SISldependent in vitro translation system or a defined SISldependent in vitro translation initiation reaction. Experlmental Procedures Yeast Strains The yeast strains used for this study are shown in Table 1. Yeast cultures were grown, as indicated, using either synthetic complete medium containing 2% glucose or YEPD medium containing 2% glucose (Sherman et al., 1989). Isolation of sosl.1 From 20 colonies. 20 independent cultures of strain CY873 (MATa [&l-85 on a URAICEN plasmid] dsisl::H/S3) were prepared in YEPD medium and grown at 3OOC. The next day, 0.1 ml of each culture was spread onto a separate YEPD plate and grown at 38.3OC. After 5 days at 38.3”G three spontaneous Ts+ revertants were picked from each plate. One of the best Ts+ revertants was pursued further and was crossed to strain CY732 (MATa [sisl-85 on a LfUICEN plasmid] dsisl::H/S3). The progeny from this cross showed that the temperature-sensitive phenotype segregated 2 Ts+/2 Ts, indicating that a single nuclear mutation gives rise to the Ts+ suppression, that the Ts+ suppressor mutation was unlinked to URA3 (which is linked to the original sisl-85 gene, showing that the mutation giving rise to the Ts+ phenotype was not within the original sis7-85 gene), and that the Ts+ phenotype was 100% linked to a slow growth phenotype that, like the Ts’ phenotype, segregated 212 (indicating that the Ts+ suppressor mutation causes a slow growth phenotype). We term this suppressor mutation sosl-1. The sosl-7 mutation wascrossed into awild-types/S7 genetic background. Ten plasmids that complement the growth defect of a sosl-1 strain were isolated from a YCpBO yeast genomic library (Rose et al., 1987). Restriction enzyme analysis showed that all of these plasmids contained a common region of yeast DNA. A region of the yeast insert of one of these plasmids was subcloned into the IMA3 integrating vector Ylp5. The resulting plasmid was cut within yeast DNA sequences and transformed into a wild-type SOS1 ufa3-7 strain, which

Cell 1164

Table

1. Yeast

Strains

Strain

Genotype

Source

AY925 AY926 CY457 CY732 CY733 CY673 CY1741 CY 1746 CY2012 CY2107 CY2194 CY2195 CY2196 CY2509 CY2514 CY2519 CY2522 CY2952

W303: Same AY925 AY925 AY925 AY926 AY926 AY925 AY926 AY926 MATa MATa MATa AY926 AY925 MATa MATa MATa

R. Rothstein’ Ft. Rothstein” Luke et al. (1991) Luke et al. (1991) Luke et al. (1991) This study This study This study This study J. Warnerb YASlOO of Sachs and Davis YASl20 of Sachs and Davis YAS262 of A. SachsC This study This study F96 of A. Hinnebuschd F294 of A. Hinnebuschd J771-44A of J. Warned

MATa ura3-1 /eu2-3, 112 his3-11, 15 trpl-7 ade2-1 ssdl-d2 canl-100 as AY925 but MATa plus Asisl::H/S3 (NH,-HA-tagged wild type SW on LEU?/CEN plasmid) plus Asisl::H/S3 (NHrHA-tagged sisl-85 on LEtJ2K.fN plasmid) plus Asisl::H/S3 (NHrHA-tagged sisl-86 on LEKVCEN plasmid) plus Asisl::H/S3 (NH*-HA-tagged sisl-85 on YCp50) plus Asosl::LEU2 plus Asosl ::LEUP plus Asosl ::LEU2 Asisl::H/SS (NHrHA-tagged sisl-85 on YCp50) plus Arpl4a::URA3 Apabl::H/S3 ura3 leu2 his3 trpl ade2 (PA61 on URA3/cen plasmid) Apsbl::HIS3 wad leu2 his3 trpl ade2 (pabVF364L on TRPl/CEN plasmid) AspbZ::LEU2 wad leu2 his3 Atrpl plus AsosP::Uf?AS plus Arpl4a::URAS gcdl-101 ura3-52 prtl-1 ura3-52 /eu2-3, 112 adel Arps28B::LYSP ura3-52 /eu2-1 his3-61 trpl-a /ys2-8136 met&l

(1969) (1989)

Columbia University, New York, New York bAlbert Einstein, Bronx, New York ‘University of California, Berkeley, California ONational Institutes of Health, Bethesda, Maryland

directs integration of URAB at the SOS7 locus. A Ura’ transformant was crossed to a sosl-1 wad-7 strain and shown by tetrad analysis to have URAI integrated at the SOS1 locus. (In 17 tetrads total, 15 tetrads gave 2 Ura- slow growing colonies plus 2 Ura’ colonies with a normal growth rate, and 2 tetrads gave 1 Ura- slow growing colony plus 2 Ura’colonies with a normal growth rate.) Therefore, the complementing piasmid contained the authentic SOS1 gene. The URAS marked SOS7 locus was also used to show that suppression of the sisl-85 mutation was 100% linked to the sosl-1 mutation. Isolation of SOS2 Southern analysis of a Asosl strain showed that yeast cells contain an additional DNA region that hybridizes to the SOS1 probe at high stringency. This region was isolated by probing a YCpSO yeast genomic library (Rose et al., 1987) with DNA sequences +506 to +776 of SOS1 (a Hindlll fragment of SOSl). The DNA sequence of the SOS2 coding region is very similar to that of SOS1 (only three base changes over a 357 nt region). In addition, the SOS1 intron has limited sequence similarity to the intron of SOS2. DNA Sequencing SOS1 and SOS2 were cloned into pUCll8 vectors in both orientations. Unidirectional deletion series were prepared for each orientation. The SOS7 and SOS2 genes were sequenced completely on both strands using the Sequenase enzyme system (US Biochemical). Chromosomal Deletions of SOS1 and SOS2 The SOS1 and SOS2 genes each have two Hindlll sites located at identical positions within the coding sequences (corresponding to amino acids 7 and 97 of SOS1 and SOS2). For SOSI, DNA sequences between the two Hind81 sites were replaced with a 2.2 kb Sall-Xhol DNA fragment containing the LEUP gene. For SOS2, DNA sequences between the two Hindlll sites were replaced with a 1.2 kb Hindlll fragment containing the URAB gene. The Asosl::LEU2 and the Asos2:: URA3 chromosomal mutations were confirmed by Southern analysis. The SOSf and SOS2 Genes Are Located on a Duplicated Region of Chromaaome IV When a Asosl::LEU2 strain is crossed to a Asos2::URA3 strain, 6 parental ditype tetrads (2 Leu+ Ura- and 2 Leu- Ura’ colonies per tetrad), 8 tetratype tetrads (1 Leu’ Ura-, 1 Leu- Ura’, and 1 Leu Uracolony per tetrad), and no nonparental ditype (we would expect 2 Leu’ Ura’ and 2 Leu- Ura- colonies per tetrad) were obtained in 14 tetrads total. Therefore, SOS1 and SDS2 are about 28 CM apart. The DNA

sequence of a SOSl-containing DNA fragment shows that the predicted AUG start codon of SOS1 is located 800 bases from the stop codon of ARFl (both genes transcribed in the same orientation), which is 1 of 2 genes that encode ADP-ribosylation factor (Stearns et al., 1990). The SOS2 gene is located within about 10 kb of ARF2, as determined by hybridizing a SOSZ-specific probe to a h phage library of yeast inserts (Olson et al., 1986). The ARF2 gene is located about 28 CM from ARfl on the left arm of chromosome IV and encodes a protein that is 96% identical to the ARFl protein (Stearns et al., 1990). Like the SOS1 gene, the PPH2alPPH22 gene is located very close to ARFl. (PPH2alPPH22 is 16.7 CM from CDCQ [Sutton et al.. 19911, and ARFl is 16.2 CM in the same direction from CDCQ [Stearns et al., 19901.) The PPH2fl/PPH27 gene, which encodes a protein that is 98% identical to the PPH2alPPH22 protein over most of its length, is located about 35 CM from PPH2alPPH22 in the direction of ARF2 (Sneddon et al., 1990). Therefore, PPH2BIPPH27 is close to ARF2. From a comparison of the two members of each a gene pair (SOS1 versus SOS2, ARFl versus ARf2, and PPH2alPPH22 versus PPH~JY PPH21), the DNA sequence of the open reading frames, including the third base of the codons, is found to be very similar. Therefore, the region of chromosome IV containing SOSl, ARFl, and PPH2alPPH22 and the region of chromosome IV containing SOS2. ARf2, and PPH281 PPH27 may represent a duplication. It remains to be determined whether other genes are present as duplicated copies in this region of the left arm of chromosome IV. Sucrose Gradients Analysis Yeast cells from a 100 ml culture were lysed by gentle vortexing in the presence of glass beads. This mixture was centrifuged at 13,000 x g for 10 min at 4°C. Then, 25 ODm units (about 300 ul) of the supernatant (i.e., extract) was loaded onto 10 ml sucrose gradients. The tubes were centrifuged at 39,000 rpm for the indicated amount of time in a Beckman Instruments SW41 rotor at 4’C. The gradients were dripped through a UA-6 ultraviolet monitor (lsco) and monitored at 254 nm. For the Western analysis, fractions were collected directly from the flow cell. The fraction collector was connected to the ultraviolet monitor so that each tube change of the fraction collector was indicated on the recorder output. For the sucrose gradients in Figures 4. 5, and 6, cycloheximide was added to the culture (to 60 uglml final concentration) just prior to harvesting the cells (except for Figure 58, in which the cycloheximide was added just prior to shifting the culture to 37°C). The extracts were prepared in a buffer consisting of 10 mM Tris (pH 7.4) 100 mM NaCI. 30 mM MgCl*, 60 uglml cycloheximide, 200 uglml heparin (ex-

7;::

and the Initiation

of Translation

cept for Figure 5C, in which the NaCl concentration was increased to 0.6 M). The extracts were loaded onto linear 7%-47% sucrose gradients prepared in 50 mM Tris-acetate (pH 7.0) 50 mM NH&I, 12 mM MgCl*, 1 mM dithiothreitol buffer (except for Figure 5C, in which the NaCl concentration was increased to 0.7 M). The gradients were centrifuged for 2.5 hr. The horizontal and vertical scales are identical between Figures 4. 5, and 6. For the sucrose gradients in Figure 7, cycloheximide was added to the cultures (to 100 pg/ml final) just prior to harvesting the cells. The extracts were prepared in a buffer consisting of 20 mM HEPES (pH 7.5). 10 mM KCI, 5 mM MgCI,, 1 mM EGTA, 100 mglml cycloheximide, and 2 mM dithiothreitol. The extract used for Figure 78 was treated with RNAase A (Sigma; stock solution at IO mg/ml boiled for 15 min and extensively dialyzed into the extract preparation buffer) at a final concentration of 300 uglml. The extracts were loaded onto linear 20%47% sucrose gradients prepared in the same buffer used for the extract preparation, except that no dithiothreitol was present. The gradients were centrifuged for 3.5 hr. For the Western analysis, 0.6 ml fractions were collected (except for Figure 7E, in which 0.4 ml fractions were collected). The proteins in each fraction were precipitated by addition of 50% trichloroacetic acid (to 12.5% final concentration), followed by incubation overnight at -2OOC. The proteins were pelleted by spinning in a microfuge for 15 min at 4OC. The pellets were washed twice with 1 ml O°C acetone and resuspended in protein gel loading buffer. Onehalf of each fraction was IoadedontoaSDS-polyacrylamide(lO%)gel. Blots of the gels were probed for SISI with #252 antiSIS polyclonal antiserum (Luke et al., 1991) using an ECL detection system (Amersham Corporation). Acknowledgments We thank A. Sachs forpabl and spb2 strains, A. Hinnebusch for gcdl and prtl strains and advice on sucrose gradients, J. Warner for the rpl4a and rps28b strains, A. Caplan for communication of results prior to publication, members of the Arndt lab for discussion, and A. Sutton, M. Luke, D. Shevell, H. Ma, T. H. Ni, and B. Futcher for comments on the manuscript. This research was supported by National Institutes of Health grant GM45179 to K. T. A. Received

December

23, 1992; revised

March

19, 1993.

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Acceeeion

Number

The accession numbers for the SOS1 and SOS2 sequences in this paper are M82913 and L02328, respectively.

reported