Proc. Natl. Acad. Sci. USA Vol. 91, pp. 6880-6884, July 1994 Cell Biology Yeast Srplp has homology to armadillo//,- and participates in apparently multiple nuclear functions including the maintenance of the nucleolar structure RyonI YANO*, MELANIE L. OAKES, MICHELLE M. TABB, AND MASAYASU NOMURA Departments of Biological Chemistry, and Microbiology and Molecular Genetics, University of California, Irvine, CA 91717-1700 Contributed by Masayasu Nomura, April 7, 1994

ABSTRACT SRPI, a suppressor of certain temperature- MATERIALS AND METHODS sensitive mutations in RNA polymerase I in Saccharomyces cerevisuae, encodes a protein that is associated with nuclear Synthetic glucose medium is 2% glucose, 0.67% Bacto yeast pores. By using a system of conditional SRPI expression and nitrogen base (Difco), and 0.5% Casamino acids (Difco), which was supplemented with tryptophan and required by isolating temperature-sensitive srpl mutants, we have bases, as described (6).-Synthetic galactose medium is the demonstrated that is essential for maintenance of the Srplp same as above, except that 2% galactose is substituted for crescent-shaped nucleolar structure, RNA transcription, and glucose. the proper functions of microtubules as inferred from anal- Yeast strains NOY477 and NOY481 are both Mata ade2-1 ysis ofnuclear division/segregation and immunofluorescence ura3-1 his3-11 trpl-1 leu2-3,112 canJ-100 Asrpl::LEU2 and microscopy of microtubules. Different mutant alleles showed carry pNOY134 and pNOY138, respectively. pNOY134 is a significantly different phenotypes in relation to these appar- derivitive of pRS316 (7) carrying SRPI (1). pNOY138, which ently multiple functional roles of the protein. We have also carries SRPI under the control of the GAL7 promoter, was found that eight imperfect 42-amino-acid tandem repeats constructed in the following way. The HindIII-Bgl II frag- present in Srplp are similar to the 42-amino-acid repeats in ment carrying the GAL7 promoter was cut out from pAA7 (8), armadillo/plakoglobin/jJcatenin proteins present in adhe- inserted into pUC19 between the HindIII and BamHI sites, sive junction complexes ofhigher eukaryotes. We discuss this and then recovered as a HindllI-Sma I fragment. The similarity in connection with the observed pleiotropic effects Sau3AI-Xba I fragment carrying the 5' part ofSRPI (starting of srpl mutations. from -63; + 1 is A of the initiation codon) and the Xba I-Bgl II fiagment carrying the 3' part of SRPI were cut out from The SRPJ gene in Saccharomyces cerevisiae was originally pNOY134 and inserted separately into pUC19 between the identified as a specific suppressor (SRPI-1) oftemperature- BamHI and Xba I sites and between the Xba I and BamHI sensitive (ts) mutations in the zinc-binding domain of the sites, respectively. The 5' and the 3' parts ofSRPI were then A190 subunit of RNA polymerase I (Pol I) (1). The sup- recovered as a Sma I-Xba I fragment and a Xba I-Sma I pressor, SRPJ-1, was also found to suppress ts mutations in fragment, respectively. These three fiagments, the GAL7 the zinc-binding domain ofthe A135 subunit of Pol I but not promoter and the 5' SRP1 and the 3' SRP1 fiagments, were other ts mutations in both A190 and A135. There is evidence then inserted into the multicloning sites of pRS316 using the to suggest that the zinc-binding domains of the two poly- HindIII, Sma I, and Sac II sites, yielding pNOY138. merase subunits, one close to the N terminus of A190 and For isolation of srpl ts mutants, pNOY162 DNA, which the other close to the C terminus of A135, are in physical carries TRPI and SRPI (see below), was treated with hy- proximity and interact with each other (2). It was therefore droxylamine (9) and then transformed into NOY510 [same as originally inferred that the SRPI gene product interacts NOY481, but carries pNOY137 (URA3, SRPJ, 2,u)] with directly or indirectly with this region of Pol I (1). The SRPI selection for tryptophan at 30'C. Tryptophan-positive trans- gene was cloned and sequenced, and the encoded protein formants were then subjected to a plasmid shuffle screening was immunofluorescence procedure (10) using 5-fluoroorotic acid to counterselect cells (Srplp) localized, by microscopy carrying pNOY137 and using 250C and 380C as permissive as well as biochemical fractionation, to the periphery ofthe and nonpermissive temperatures. In this way, mutant plas- nucleus, in the vicinity of the nuclear pores (1). This mids carrying srpl-31, srpl49, and srpl-54 (pNOY163, localization, which was subsequently confirmed by addi- pNOY166, and pNOY168) were isolated, and altered amino tional experiments (mentioned below), was surprising in acids were identified by DNA sequencing. The mutant and view of the specific genetic interaction between SRPI and the control plasmids were then transformed into diploid strain the Pol I subunit genes mentioned above. In this paper, we NOY476 (1) and sporulated, and haploid segregants with the first describe the results of mutational analysis of SRPI as chromosomal Asrpl: :LEU2 and carrying the SRPI as well as well as Srplp depletion experiments. We then describe our srpl mutant plasmids were recovered. The four strains ob- finding of structural similarity of Srplp to a family of tained in this way were transformed with pRS316 to make proteins including the vertebrate adhesivejunction proteins them uracil-positive, thus yielding a set of the four strains, f3-catenin and plakoglobin (3) and the Drosophila segment NOY525, NOY521, NOY522, and NOY523, used to study polarity-determining protein armadillo (4, 5) and discuss effects of the mutations. [To construct pNOY162, the Hpa possible functions of Srplp in relation to the observed I-Bgl II 2.4-kb fragment carrying SRPI was first inserted into pleiotropic effects caused by mutations in this protein or by Sma I-BamHI sites of pUC19. SRPI was then recovered its depletion. Abbreviation: DAPI, 4',6-diamidino-2-phenylindole; ts, temperature sensitive; Pol I, RNA polymerase I. The publication costs ofthis article were defrayed in part by page charge *Present address: Laboratory for Neural Networks, Frontier Re- payment. This article must therefore be hereby marked "advertisement" search Program, The Institute of Physical and Chemical Research in accordance with 18 U.S.C. §1734 solely to indicate this fact. (RIKEN), Wako, Saitama, 351-01, Japan. 6880 Downloaded by guest on October 1, 2021 Cell Biology: Yano et al. Proc. Natl. Acad. Sci. USA 91 (1994) 6881 from this construct as the Kpn I-Sal I 2.4-kb fragment and A SSB1 L)NA inserted into Kpn I-Sal I sites of pRS314.1 For isolation of suppressors of the rpal90 (and rpal35) ts mutations, pNOY162 DNA mutagenized as described above was introduced into NOY267 carrying the rpal9O-5 mutation (11), and plates were incubated at 25TC for 2 days followed by an additional 1-2 days incubation at 37C. Plasmids were I recovered from transformants that could grow at 37TC, and their ability to allow NOY267 to grow at 3rC was examined by repeating transformation. The plasmids carrying suppres- sor mutations were then subjected to DNA sequence analy- sis, and altered amino acids were identified. RESULTS Effects of Srplp Depeto on Growth, rRNA Synthesis, and Nudeolar Structres. We constructed a strain (NOY481) in which transcription of the SRPI gene was controlled by the inducible GAL7 promoter. The strain grew on galactose but not on glucose plates, confirming the conclusion (1) that SRP1 B DNA DIC is an essential gene required forgrowth. NOY481 and a control strain, NOY477, were first grown in galactose medium and then transferred to glucose medium. Cell growth and [3H]uri- dine incorporation activity were followed. About 10 hr after transfer to glucose medium, a decrease in the growth rate of I NOY481 became noticeable. At this time the cellular concen- FIG. 1. Immunofluorescence of nucleolar proteins and DAPI tration ofSrplp was about 10% ofthat in the control strain as staining of DNA in NOY481. Cells were first grown in synthetic analyzed by Western immunoblot. The [3H]uridine incorpo- galactose medium and then shifted to synthetic glucose medium. Cell ration activity ofthe experimental (NOY481) culture started to density was kept below 0.8 by dilution of the cultures. (A) At 0, 10, decrease sharply at some time between 8 and 10hr. RNADNA and 14 hr after the shift, the cells were analyzed by immunofluores- hybridization analysis using an rRNA-encoding DNA probe at cence ofthe nucleolar protein SSB1 (rhodamine) and DAPI staining 8 and 12 hr showed that the synthesis of not only rRNA but of DNA as described (12). (B) Cells at 10 hr after the shift were also was analyzed by double-label immunofluorescence staining of the Pol I also, to a lesser extent, other RNAs affected by Srplp A190 subunit (fluorescein) and SSB1 (rhodamine). DAPI staining of depletion, confirming an eventual nearly complete inhibition DNA and Nomarski differential interference contrast (DIC) pictures of total transcription (data not shown). of the same cells are also shown. (Bar = 5 um.) We note that the In normal yeast cells, the nucleolus can be seen as a length ofa protruded loop (e.g., see DNA inB) is 6-8 pAm. Ifa single crescent or oval structure by immunofluorescence micros- loop represents an rRNA-encoding DNA cluster that would be about copy with suitable nucleolus-specific probes, such as anti- 370 pm iffully extended (9.1 kb per unit repeat times =120 repeats), bodies against Pol I subunit A190, fibrillarin, or SSB1 (a then the calculated packing ratio of DNA in this region would be single-stranded nucleic acid-binding protein). In Srplp de- about 50, which is comparable to the packing ratio of DNA in the pletion experiments, we found that at 8 and 10 hr after the 30-nm chromatin fiber (about 30). In a significant fraction of cells, stained with anti-A190 protrusion of two DNA loops from two nearby locations in a single shift to glucose, the nucleolar region nucleus were seen, giving a Mickey Mouse-like morphology, but in or anti-SSB1 has a markedly different morphology ("unfold- some other cells only a single protusion was observed. We think that ed") and becomes clearly separated from the main body of the nuclei with two protrusions are probably in the 2n stage (G2/M the round nucleus stained with 4',6-diamidino-2-phenylin- stage in cell cycle); each protruded DNA thread corresponds to the dole (DAPI; Fig. 1). In many cases, we can see fibrous single rRNA cluster on one of two copies of chromosome XII. threads (often seen as loops) stained with DAPI that protrude from the main body of the nucleus, and nucleolar proteins Clark, and M.N., unpublished experiments). Therefore, the (SSB1 and Pol I) stained with antibodies are located along morphological change in the nucleolus does not appear to be these "DNA threads" (see Fig. 1B and nuclei with arrow- caused by gross damage to the nuclear pores. heads in Fig. 1A). We presume that these DNA threads, We have also observed that, upon further depletion of which are sometimes clearly seen as a loop, contain the Srplp, nuclear division and/or segregation becomes defec- rRNA gene cluster, but this has not been proven (see tive. The frequency of cells showing obvious defects in comments in Fig. 1 legend). proper nuclear segregation (large-budded cells with the nu- Upon further incubation in glucose, nucleoli were frag- cleus only in one of the paired cells and single cells without mented, and the fragments appeared to be distributed any nucleus) increased from 1% at time 0 to 30%o or more of throughout the nucleus, but not in the cytoplasm (14 hrin Fig. the total cell population at 14 hr. Although the terminal 1A). A similar fragmented structure called "'mininucleolar morphological phenotype was not uniform, suggesting prob- bodies" was observed in cells with mutationally inactivated able pleiotropic effects of depletion, it is evident that Srplp Pol I (12). Thus, Srplp appears to play an important role in is required, directly or indirectly, to achieve proper nuclear the maintenance ofthe crescent nucleolar structure. It should division/segregation. be noted that the morphological alterations of the nucleolus Isolation of srpl Mutations That Are ts or Suppressors of were not accompanied by gross alterations of the nuclear rpal9O ts Mutations. We isolated srpl ts mutants. Three envelope. First, we do not see leakage into the cytoplasm of mutants that showed single-base substitutions (srpl-31, srpl- nucleolar proteins, such as SSB1, fibrillarin, or Pol I sub- 49, and srpl-54) were selected for further studies. We also units, during the times of analysis. Second, immunofluores- isolated SRPI mutants carrying suppressor mutations on the cence microscopy using antibodies (Mab306) against nucle- plasmid that allow growth of rpal90-5 mutants at nonper- oporins and electron microscopic examination of Srplp- missive temperatures. In addition to the SRPI-I allele studied depleted cells (at 10 hr) showed no visible alterations in the previously (1), two additional alleles, SRPI-2 and SRPI-3, nuclear envelope including the nuclear pores (M.L.O., M. were identified. alterations caused by these Downloaded by guest on October 1, 2021 6882 Cell Biology: Yano et al. Proc. Natl. Acad. Sci. USA 91 (1994) mutations are shown in Fig. 2. As reported in the previous DNA Tubulin paper (1), Srplp contains eight imperfect, 42-amino-acid A tandem repeats in the center of the molecule. Except for srpl-31, all the ts and suppressor mutations are at sites within the central domain containing the repeats (Fig. 2; see the legend). Phenotypes of spl) ts Mutations. We studied a set of four strains carrying -SRPJ, srpl-31, srpl49, or srpl-54 on a centromere plasmid and having the chromosomal SRP1 gene disrupted. Both srpl-31 and srpl-S4 mutants showed a re- B duced growth rate (both 70-80%o of the control) and de- creased [3H]uridine incorporation activity (both 20-30%6 of the control) even at 25TC. After a temperature shift to 380C, their growth rate as well as [3H]uridine incorporation rates further declined only very slowly. In the case ofsrpl49, both the growth rate and the [3H]uridine incorporation rate were about the same as the wild-type strain at 250C and up to 4 hr after the shift to 38TC. A slow decline in growth rate accom- FiG. 3. Immunofluorescence staining ofmicrotubules (a-tubulin) panied by relatively faster decline in [3H]uridine incorpo- and DAPI staining ofDNA in SRPI (A) and srpl-49 (B) cells 6 hr after ration rate started thereafter, reaching a [3H]uridine incor- shift to 38(2. Arrowheads show an example of extended microtu- rate 200% of the wild at 8 hr after the bules connecting two separated daughter nuclei in SRPI cells. poration of about type Arrows show an example of large-budded cells containing the main shift (data not shown). nuclear body in one cell and the (unfolded) nucleolus in the other cell Regarding nucleolar morphology, the srpl49 allele showed and with bipolar microtubules clearly misoriented. (Bar = 5 pum.) clear changes, starting at =4 hr after a temperature shift to 380C. The "nucleolus" revealed by immunofluorescence mi- srpl49 cells, which had large buds and failed in nuclear croscopy using anti-SSB1 or anti-Pol I was seen to be sepa- division/segregation, the spindle pole bodies had duplicated rated clearly from the main nuclear region revealed by strong and nuclear microtubules connecting the two spindle pole staining with DAPI, and this feature was seen in a majority of bodies were apparent. In a significant fraction (-2096) ofthose cells at 6 hr after the shift. In addition, unfolded thread-like cells with extended microtubules, the two ends were localized nucleolar structures resembling those seen upon Srplp deple- away from the bud neck so that the microtubules were nearly tion were also observed (10-20%6 of the total population; e.g., perpendicular to the bud axis, indicating mutational defects in see large-budded cell with an arrow in Fig. 3B). Defects in the ability to orient microtubules along the bud axis (Fig. 3B). nuclear division/segregation similar to those seen upon Srplp In addition, a significant number (about 50%o) of unbudded depletion were also clearly observed for this mutant; at 8 hr srpl49 cells had an extended bipolar nuclear microtubule, after the shift, =42% of the total cell population displayed a whereas most of the unbudded cells in the control wild-type nuclear segregation-defective phenotype. culture showed single stained dots representing a nondupli- In contrast to srpl49, both the srpl-31 and srpl-54 alleles cated spindle pole body and did not show any clearly recog- showed either no change or only a slight change in the nizable bipolar nuclear microtubules (<1%). Such abnormal nucleolar morphology after shifting to nonpermissive tem- features as well as other aberrant morphologies were, com- peratures. Defects in nuclear division/segregation were not pared with srpl49, more pronounced in Srplp-depleted cells clearly observed either. at later stages, less marked for srpl-31, and not observed for The defects in nuclear division/segregation might be srpl-S4 at nonpermissive temperatures. caused by defects in microtubule-related functions. Regard- Sequence Similarity of Srplp to Armadiilo/Plakoglobin/f- ing this question, in collaboration with us, D. Pellman (per- Catenin Family Proteins. We have now found that the 42- sonal communication) examined the chromosome instability amino-acid repeats in Srplp we reported earlier (1) are similar of the three srpl mutants described here by assaying the loss to the 42-amino-acid repeats that were originally discovered of a chromosome fragment containing SUP)) in an ade2-201 in the protein encoded by the Drosophila segment polarity strain. A large increase (about 20-fold) in the frequency of gene armadillo (4). The armadillo protein was subsequently chromosome loss was observed for srpl-31 (at 30°C, a shown to be a homologue of the vertebrate adhesivejunction semipermissive temperature) but not for srpl49 and srpl-S4. proteins plakoglobin and ,3-catenin (4, 5). In Fig. 4, we show It appears that the two different alleles, srpl]49 and srpl-31, the eight 42-amino-acid repeats and the "SRP1 consensus cause related but distinct phenotypes. sequence" as published in the earlier paper (ref. 1, see the By immunofluorescence microscopy using anti-tubulin an- legend to Fig. 4) and compare them with the armadillo tibodies, we directly examined the state ofmicrotubules in the consensus sequence (ref. 4; see the legend to Fig. 4). It is three srpl mutants at the nonpermissive temperature, as well clear that the SRP1 repeats match well the armadillo con- as in Srplp-depleted cells. We found that in many of the sensus sequence and vice versa; that is, the structure of the central domain of Srplp is similar to that of the armadillo srpl-31 srpl-49 srpl-54 S116F E145K G459V protein in terms of the number (42) and sequence of amino + t srp1-1 srpl-3 srpl-2 acids in each repeat. Alignment of the complete protein P219Q D286N E360K t sequences using the Genetics Computer Group program GAP 200 (15) also confirmed the above conclusion. .. 1001 ...... 1300II t 400 500 rT,,T t r II I II __ IlI_ DISCUSSION FIG. 2. Locations of various srpl mutations and their amino acid alterations. For example, S116F indicates alteration of serine at Srplp Plays a Role in the Maintenance of Nucleolar Struc- position 116 to phenylalanine. The positions of eight 42-amino-acid ture. We have observed striking morphological changes in the repeats are indicated as boxes with arrows. We note that because of nucleolus upon Srplp depletion as well as after an upward the ambiguity of the exact start site of the first repeat (see the legend temperature shift of srpl49 mutant cells, but such changes to Fig. 4) srpl-31 could also be in the domain with tandem repeats. were not apparent for srpl-31 and srpl -54 mutants. The initial Downloaded by guest on October 1, 2021 Cell Biology: Yano et aL Proc. Natl. Acad. Sci. USA 91 (1994) 6883

N D L CONSENSUS ARN KAI----GGIPALVRLL-R--N---AL-AA--VLHNLS-----N SCORE (%) CONSENSUS ELA VQE K S S VT T R A S ARM SRP1 SRP1 (123) IDVVIQAGVVPRLVEFNREN-OPENLOLEAAWALTNIASGTSAQ 38 86 REPEATS (166) TKVVVDADAVPLFIILLYTG-SVE-VKEIAIWALGNVA6DSTDY 38 73 (208) RDYVLOCNANEPILGLFNSN-KPS-LIRTATWTLSNLCRGKKPQ 31 64 (250) PDWSVVSQALPTLAKLIYSN-DTE-TLVDACWOkISYLSDGPOEA 35 64 (292) IOAVIDVRIPKRLVELLSHE-STL-VQTPALRAVGNIVT6NDLQ 35 68 (334) TQVVINA6VLPALRLLLSSP-KEN-IKKEAOCTISNITAGNTEO 38 82 (376) IOAVIDANLIPPLVKLLEVA-EYK-TKKEACWAISNASSG66LR 42 82 (418) PflLVSOGCIKPLCDLLEIA-DNR-IIEVTLDALENILKNGEAD 31 50 IIR Av. 36 Av. 71 L LL L L L L SRP1 -D-VI-A-IIP-LI-LL------I---A-WAISNIA-G---Q CONSENSUS V VY V V VYS ARN 12 REPEAT SEQUENCES (RIGGLEMAN ET AL., 1989) 64, 68, 80 50, 36, 59 REPEATS NOT SHOWN 64, 68, 60 41, 36, 36 48, 72, 48 45, 36 41 32. 60. 56 27. 41. 32 Av. 60 Av. 40 FIG. 4. Comparison of42-amino-acid tandem repeats in Srplp with the tandem repeats in armadillo protein. The consensus sequence defined for armadillo (ARM; taken from ref. 4) and that defined for Srplp (SRP1; taken from ref. 1) are given. Amino acids that match consensus sequences in each repeat are counted and divided by the number of residues defined in a consensus sequence. Percentage values obtained in this way are shown as the "consensus score." A half repeat in armadillo (4) is omitted here. Note that the original armadillo consensus starts with G at position 8 (indicated by an arrow). The SRP1 consensus includes amino acids at positions 9 (L, I, V), 14 (L, I, V), and 38 (A, S) that were not included before (1). L, I, and V as well as A and S are grouped as similar amino acids. In addition, since the armadillo consensus sequence defined was 44 amino acids long, which allows two gaps for individual armadillo repeat sequences for good alignment (4, 13), the SRP1 consensus given here is also made 44 amino acids long by adding a gap after the 20th amino acid to all the Srplp repeats. Note also that all the repeats of Srplp are 42 amino acids long except for the first (43 amino acids) and last (45 amino acids) repeats. We also note that the Genetics Computer Group program Go (with default setting) gave 17% amino acid identity and 43% similarity between armadillo and Srplp, whereas comparing only the repeat region sequences of the two proteins gave 20% identity. Similar computer analysis gave 23% identity and 49%o similarity between Srplp (542 amino acids) and human plakoglobin (743 amino acids; ref. 14). morphological change of the nucleolus unfolding observed functions (17). These observations also suggest that Srplp upon Srplp depletion appears to take place before or simul- plays important roles in microtubule-related functions. taneously with the onset of significant inhibition of RNA Srplp May Play a Regulatory Function Similar to Aadil- synthesis. We believe that the inhibition of rRNA synthesis lo/Plakoglobin/3-Catenin. A salient feature of our observa- is not the cause ofthe morphological change ofthe nucleolus tions on Srplp regarding its functions is its apparent partic- but rather a consequence of the change of the nucleolar ipation in several distinct nuclear functions, such as the structure. This conclusion can be supported by the results of maintenance of the intact crescent-shaped nucleolar struc- our experiments in which the nucleolar morphology was ture and regulation of proper function of microtubules, as examined by using various RNA synthesis inhibitors such as discussed above. In addition, it is now established that Srplp 3-aminotriazole or conditions of decreased rRNA synthesis interacts with the nuclear pore proteins, Nupip and Nup2p, such as carbon starvation. These treatments failed to produce functionally and physically. Synthetic lethality was demon- not only the pattern of unfolding but also the pattern of strated by combination of srpl with nupl (L. I. Davis, fragmentation observed in later stages of Srplp depletion personal communication) as well as with nup2 mutations (12). Thus we conclude that Srplp plays an essential role, (J. D. J. Loeb, personal communication). Thus, Srplp may directly or indirectly, in the maintenance of the nucleolar also participate in functions carried out by these porins. structure, and also suggest that efficient rRNA transcription Srplp was originally localized to nuclear pores by immuno- requires proper nucleolar structures. fluorescence microscopy (1), and, subsequently, physical Srplp Plays a Role in Microtubule-Related Functions. We interactions of Srplp with Nupip and with Nup2p were have found clear defects in nuclear division/segregation in directly demonstrated by a variety of methods including one (srpl49) ofthe srpl ts mutants as well as in cells depleted coimmunoprecipitation of these two porin proteins with of Srplp. It has been shown that faithful nuclear segregation Srplp (L. I. Davis, personal communication). Therefore, the requires a correct orientation of mitotic spindles before the observed synthetic lethality between srpl and nupi or nup2 onset of anaphase, which in turn requires proper functioning may not be surprising. However, the apparently specific of cytoplasmic microtubules including their (indirect) inter- involvement of Srplp in microtubule-related functions, nu- action with filaments (16). Indeed, direct examination of cleolar structure maintenance, and Pol I function (or assem- microtubules in srpl49 and Srplp-depleted cells revealed bly or stability) are difficult to explain by direct involvement aberrant morphology of microtubules. The increase in the of Srplp itself in these diverse functions. Two alternative chromosome loss observed for another allele, srpl-31, is a models can be considered. First, Srplp plays a direct role in distinct phenotype, but it may also be due to defects in a transport of macromolecules through nuclear pores, and all microtubule-related function. the pleiotrophic effects of srpl mutations as well as Srplp The SRPI gene was independently isolated in a screen for depletion are consequences of effects on transport. For mutations showing synthetic lethality with biki (D. Pellman example, suppression ofcertain rpal90 and rpal35 mutations and G. Fink, personal communication) and as a high-copy- in the zinc-binding domains by SRPI suppressor mutations number suppressor gene that remedies cold sensitivity and might be due to increased transport of the A190 or A135 defects in chromosome segregation caused by a csel mutation subunits, compensating for (presumed) instability of the (Z. Xiao and M. Fitzgerald-Hayes, unpublished experiments mutant forms of these subunits. [Based on the different cited inref. 17). The protein encoded byBIKI is amicrotubule- pattern of suppression between the SRP1 suppressor associated protein that is colocalized with tubulin in the (SRPI-1) and another suppressor, SRPS, that caused sup- mitotic spindle as well as in spindle pole bodies and appears to pression by increased subunit synthesis (19), we did not favor play a role(s) in microtubule-related functions (18). CSEJ is this explanation in our earlier paper (1), but the different required for faithful chromosome segregation, and, therefore, pattern could be explained by postulating a large difference the encoded protein may also carry out microtubule-related in the degree of suppression between SRP5 and SRPI-1.1 Downloaded by guest on October 1, 2021 6884 Cell Biology: Yano et A Proc. Natl. Acad. Sci. USA 91 (1994) Similarly, defects in the maintenance of nucleolar structures with Srplp independently (L. I. Davis, personal communi- or in microtubule functions could be explained by postulating cation). It is conceivable that the Srplp complexed with mutational defects in transport of certain macromolecules Nupip and the Srplp complexed with Nup2p belong to required for these functions. The second model is that Srplp separate systems interacting with different target structures has several different functional targets separated away from or representing different transport functions and that there its primary location at nuclear pores and that Srplp interacts might be other similar systems involving Srplp or related with these target structures by direct or indirect physical molecules. Thus, different mutations or depletion of Srplp contact with the target structures, perhaps by some signal (or increased amounts caused by a high gene dosage) might transduction mechanisms. Different srpl mutations (or de- lead to different effects in various nuclear functions. Specific pletion) are proposed to affect a different subset of (or all of) genetic interactions of SRPJ with a wide variety of other the target functions, thus causing the observed pleiotropic nuclear genes, rpal90, rpal35, bikN , csel, nupi, and nup2, as effects. Although we cannot distinguish these two alternative observed independently, may reflect the presence of such models at the moment, it should be emphasized that different multiple systems, utilizing Srplp and controlling organization srpl mutations cause apparently distinct phenotypes. For of nuclear structures and functions or controlling nuclear example, the effects of srpl-31 and srpl-54 on growth rates pore transport functions. After completion of the present are similar, yet chromosome instability was observed only for work, a letter to the editor of Cell appeared, which also srpl-31. Similarly, under the conditions that give the same described a similarity between armadillo repeats and SRP1 degree of decreased growth rate (e.g., 20-30o of the control at non-permissive temperatures), only srpl49, and not repeats (22). srpl-31 or srpl-54, show marked alteration in nucleolar We thank Drs. R. F. Doolittle and W. M. Fitch for their help in morphology. Thus if all the phenotypes are consequences of protein homology search and discussion; Drs. L. I. Davis, M. altered nuclear pore transport functions, as postulated by the Fitzgerald-Hayes, M. W. Clark, D. Pellman, and J. D. J. Loeb for first model, one must also postulate that there are several communicating their unpublished results; and Drs. R. E. Steele, specific transport systems, and different mutations in Srplp S. B. Sandmeyer, J. Keener, D. Pellman, and G. R. Fink for their would affect these systems differently. The structural simi- comments on the manuscript. This work was supported by Public larity found between Srplp and proteins in the armadillo/ Health Service Grant R37GM35949 from the National Institutes of plakoglobin/f-catenin family as described in this paper are Health. M.M.T. was supported by Public Health Service Predoctoral pertinent in discussing these two models further. Training Grant GM0713419 from the National Institutes of Health. Plakoglobin/f3-catenin are components of adhesive junc- tion complexes interacting directly with cytoplasmic portions 1. Yano, R., Oakes, M., Yamagishi, M., Vu, L. & Nomura, M. of transmembrane proteins of the family and carry (1992) Mol. Cell. Biol. 12, 5640-5651. out an essential function in anchoring cytoskeletal 2. Yano, R. & Nomura, M. (1991) Mol. Cell. Biol. 11, 754-764. filaments 3. Peifer, M., McCrea, P. D., Green, K. J., Wieschaus, E. & to the junction protein complexes. In addition, proteins in Gumbiner, B. M. (1992) J. Cell Biol. 118, 681-691. this family appear to participate in transduction of signals to 4. Riggleman, B., Wieschaus, E. & Schedl, P. (1989) Genes Dev. organize and control various cellular activities in response to 3, 96-113. cell-cell contact (for a review, see ref. 20). By analogy, in the 5. Peifer, M. & Wieschaus, E. (1990) Cell 63, 1167-1178. case ofthe second model, one could speculate that Srplp may 6. Sherman, F., Fink, G. R. & Hicks, J. B. (1986) Laboratory participate in the control of various nuclear activities by Course Manual for Methods in Yeast Genetics (Cold Spring anchoring various structural components to nuclear pores Harbor Lab. Press, Plainview, NY). and thereby organizing proper nucleoskeletal structures re- 7. Sikorski, R. S. & Hieter, P. (1989) Genetics 122, 19-27. quired for these activities. For example, it has been observed 8. Yasumori, T., Murayama, N., Yamazoe, Y., Abe, A., Nogi, by several investigators that rRNA genes are associated with Y., Fukasawa, Y. & Kato, R. (1989) Mol. Pharmacol. 35, an insoluble subnuclear fraction composed of the nuclear 443-449. envelope/skeleton in various in vitro fractionation studies of 9. Sikorski, R. S. & Boeke, J. D. (1991) Methods Enzymol. 194, 302-318. mammalian cells (ref. 21 and other references therein). In 10. Rose, M. D. & Fink, G. R. (1987) Cell 48, 1047-1060. addition, most of the actively functioning Pol I was also 11. Wittekind, M., Dodd, J., Vu, L., Kolb, J. M., Buhler, J. M., bound to the similar insoluble nuclear envelope/skeleton Sentenac, A. & Nomura, M. (1988) Mo!. Cell. Biol. 8, 3997- fraction (21). It is conceivable that the maintenance of 4008. specific nucleolar structures may depend on proper nucle- 12. Oakes, M., Nogi, Y., Clark, M. W. & Nomura, M. (1993) Mol. oskeletal structures. The observed effects of mutational Cell. Biol. 13, 2441-2455. alterations or depletion of Srplp on nucleolar morphology 13. Reynolds, A. B., Herbert, L., Cleveland, J. L., Berg, S. T. & can be explained on this basis. Gaut, J. R. (1992) Oncogene 7, 2439-2445. From the analysis of a series of deletion mutants of 14. Franke, W. W., Goldschmidt, M. D., Zimbelmann, R., Muel- ler, H. M., Schiller, D. L. & Cowin, P. (1989) Proc. Natl. armadillo, Peifer and Wieschaus (5) suggested that the 42- Acad. Sci. USA 86, 4027-4031. amino-acid repeats, which are present in 12.5 tandem copies, 15. Devereux, J., Haeberli, P. & Smithies, 0. (1984) Nucleic Acids form discrete structures that are somewhat independent and Res. 12, 387-395. additive in function. It is possible that this may also be the 16. Palmer, R. E., Sullivan, D. S., Huffaker, T. & Koshland, D. case for Srplp. Mutations at different sites of Srplp causing (1992) J. Cell Biol. 119, 583-593. different phenotypes (see above) might reflect interactions of 17. Xiao, Z., McGrew, J. T., Schroeder, A. J. & Fitzgerald-Hays, Srplp with different effector molecules (or nucleoskeletal M. (1993) Mo!. Cell. Biol. 13, 4691-4702. elements). According to the second model, such interactions 18. Berlin, V., Styles, C. A. & Fink, G. R. (1990)J. CellBiol. 111, would mediate "communication" between nuclear pores and 2573-2586. the target structures, as discussed 19. McCusker, J. H., Yamagishi, M., Kolb, J. M. & Nomura, M. above. According to the (1991) Mol. Cell. Biol. 11, 746-753. first model, these different interactions would be connected 20. Kemler, R. (1993) Trends Genet. 9, 317-321. to the proposed different transport functions of the nuclear 21. Dickinson, P., Cook, P. R. & Jackson, D. A. (1990) EMBO J. pores as mentioned in the previous paragraph. In this con- 9, 2207-2214. nection it is interesting to note that Nupip and Nup2p interact 22. Peifer, M., Berg, S. & Reynolds, A. (1994) Cell 76, 789-791. Downloaded by guest on October 1, 2021