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Developmental Genetics 9:259-265 (1988)
Genes Encoding Novel GTP-Binding Proteins in Dictyostelium Stephen A. Saxe and Alan R. Kimmel
Laboratory of Cellular and Developmental Biology, National Institutes of Health, Bethesda, Maryland
We have identified a two-member gene family in the Dictyosteliurn genome and have isolated corresponding cDNA or genomic DNA recombinant clones. Analyses of these DNA sequences predicted encoded proteins of -200 amino acids with -90% sequence identity to each other. These Dictyosteliurn proteins also share amino acid identity within the GTP-binding domains in the family of G-regulatory proteins involved in cellular regulation and transmembrane signalling. Additional structural similarities are seen with members of the rus supergene family, such as m s , ml, and rho. They are similar in size (usually -200 amino acids), possess four conserved domains involved in GTP interaction and are believed to be anchored in the membrane by fatty acid modification of a cysteine residue near the carboxy terminus. More extensive identity is observed with YPTl and SEC4, two other members of this family of genes that are essential in yeast. The amino-terminal half of both Dictyosteliurn proteins is 70% identical in amino acid sequence to the YPTI and SEC4 yeast proteins with less identity continuing through the remainder of the proteins. In addition these proteins terminate in two cysteine residues that are thought to be required for membrane anchorage.
The two genes within this Dictyosteliurn family are organized differently in the genome and are differentially regulated during development. One gene is colinear in sequence with its mRNA in the protein coding region, whereas the other gene encodes a spliced mRNA. The intron-containing gene is associated with a developmentally regulated (AAC)-repeat sequence. Finally, we have shown that the expression of one of the genes is induced during development with kinetics similar to that of other (AAC),-associated genes; conversely, the expression of the second gene is repressed at a similar developmental stage.
Key words: G-proteins, gene expression, developmental regulation
INTRODUCTION
A wide variety of cellular functions appear to be regulated by proteins that interact specifically with guanine nucleotides. Many of these G-proteins are activated by binding GTP, and they possess an inherent GTPase activity that may modulate their own action. Among the best described of these proteins is the translational elongation factor EF-Tu from Escherichia coli. The amino-acid sequence of this
Received for publication March 1 , 1988; accepted April 14, 1988
Address reprint requests to Dr. Stephen A. Saxe, Laboratory of Cellular and Developmental Biology, NIDDK (6/B1- 12), National Institutes of Health, Bethesda, MD 20892.
0 1988 Alan R. Liss, Inc.
260 Saxe and Kimmel
protein is known and the sites of specific interaction with GTP have been predicted using X-ray crystallographic methods [Jurnak, 1985; la Cour et ul., 1985; McCormick et al . , 19851. Many other GTP-binding proteins have been described recently and the amino-acid sequences for most of them are known. They all possess several structural elements that are common to EF-Tu and that correlate with sites of GTP-binding or GTPase activity. Perhaps the best studied GTP-binding protein in eukaryotic cells is encoded by the rus gene. Although widely studied the exact function of the rus proteins is not yet known. Mutations that alter GTP-binding or GTPase activity in rus have profound effects on cellular properties and can cause cellular transformation in tissue culture and promote tumors in vivo. The rus proteins are characterized by their small size (usually -200 amino acids), four conserved GTP-interaction domains, an amino-terminal sequence identity conservation of >80%, and a cysteine residue at position 4 from the carboxy terminus. Recently, a number of other genes and their proteins have been identified that share some, but not all, of the properties of the rus genes. These are the rho [rus homo- log; Madaule and Axel, 1985; Madaule et ul., 19871, rul [rus like; Chardin and Tavitian, 19861, SEC4 [from yeast; Salminen and Novick, 19871, and YPTl [from yeast; Gallwitz et ul., 19831 genes. They are very similar to the rus genes in size and spacing of the GTP-interaction domains but share only 3040% amino acid identity with the true rus genes. Nevertheless, the relatedness of these genes sug- gests a rus superfamily. Another major group of GTP-binding proteins is the G,-proteins involved in signal transduction across cell membranes through interaction with cell surface-associated receptors. These G-proteins can positively or negatively regulate the accumulation of a variety of intracellular messengers. They are usually larger than the rus proteins and complex with p and y subunits to form heterotrimers. Other GTP-binding proteins are involved in protein synthesis, movement of ions, and microtubule assembly [for reviews see Gilman, 1987 and Barbacid, 19871.
The ubiquitous nature of these GTP-binding proteins and their implicated effects on signal and mechanosensory transduction suggest functions common to all eukaryotic cells. In Dictyosrelium, development is regulated by an extracellular CAMP signalling system linked to a receptor/G-protein signal transduction system. The Dictyostelium rus protein has been suggested to influence ligand induced receptor desensitization [Van Haastert et ul., 19871. However, most of the regulatory mechanisms involved in intracellular signalling during Dictyostelium development are not understood. These include the metabolism of phosphoinositides, mobilization of CA++, and transport of ions. By analogy to vertebrate systems they are likely to involve activation of regulatory proteins by GTP. The identification and isolation of the genes encoding these putative GTP-binding proteins is critical to the understand- ing of the regulation of differentiation by cellular signalling.
We have now isolated two new genes, SASl and SAS2, from Dictyostelium discoideum that code for novel GTP-binding proteins. The proteins encoded by these two genes are -90% identical in amino acid sequence with one another and most of the observed differences are located in the carboxy terminal 25 amino acids. The proteins are members of the rus superfamily; however, they differ significantly in sequence from the true Dictyostelium discoideum rus protein, whose gene has previously been identified [Reymond et ul., 19841. In fact, these proteins are more similar in sequence to two essential yeast genes, YPTI and SEC4. Although the
GTP-Binding Protein Genes 261
functions of these two yeast genes are not known, they are both required for normal growth.
The developmental expression patterns of these two Dictyostelium genes are complementary. One of the genes is expressed in growing cells and is repressed during early development in conjunction with the formation of multicellular aggre- gates. Conversely, the second gene is induced during aggregate formation and is maximally expressed during cytodifferentiation.
MATERIALS AND METHODS RNA Purification and Northern Blotting
Dictyosteliurn discoideum were developed on filters for 0-20 hours and poly(A) + RNA was purified from a portion of the cells every 5 hours [Kimmel and Firtel, 19851. RNA was separated on a 1% CH3HgOH gel [Bailey and Davidson, 19761 and blotted onto Genescreen (New England Nuclear, Boston, MA). The filter was hybridized in 50% formamide, 50.0 mM sodium phosphate (pH 6.8), 5 x SSC, 2.0 mM EDTA, 2.5 X Denhardt's [Denhardt, 19661, 200.0 pg/ml salmon-sperm DNA, 40.0 pg/ml poly A, and 32P-labeled probe at 50 "C. The probe was an RNA transcript of the protein coding region of SASI prepared by the Riboprobe Gemini System kit (Promega).
DNA Sequencing
using double-stranded DNA plasmids [Mierendorf and Pfeffer, 19871. DNA was sequenced by the dideoxynucleotide method [Sanger et ul., 19771
RESULTS AND DISCUSSION
In the course of studying a group of developmentally regulated genes from Dictyostelium, one, SASl , was identified as encoding a GTP-binding protein (see below). When a genomic DNA blot was performed it became clear that there were two members of this gene family in the genome. A Dictyostelium genomic DNA library (Ennis, unpublished) was then screened and clones derived from the second gene, SAS2, were isolated. As is seen in Figure 1, the derived amino acid sequences of the proteins indicated that the genes encode proteins of similar size, SASl coding for a 208 amino acid protein, and SAS2 coding for one of 203 amino acids. Further, the amino acid sequences of the two proteins are -90% identical with each other. Most of the divergence is near the carboxy termini.
An amino acid comparison among all known protein sequences (GENBANK data base) and these two Dictyosteliurn genes revealed significant identities within protein domains which interact with GTP. Figure 2 shows a comparison of the amino acid sequences of some of these proteins to S A S l . Four regions of sequence relatedness are observed, which are precisely those required for GTP interaction. These regions are identical in sequence for both Dictyostelium proteins. Although SASI and SAS2 differ from true rus genes, they share a number of characteristics that suggest that they are members of the rus superfamily. This is most dramatically demonstrated in the conserved spacing of the GTP-binding domains. It is clear that sequence identity is essentially restricted to these functional domains.
The Dictyostelium genes are most similar to two different yeast genes, YPTl
262 Saxe and Kimmel
SASl
SASZ
SASl
SASZ
SASl
SASZ
SASl
SASZ
SASl
SASZ
SASl
SASZ
MSPATNKSAAYDYLIKLLLIGDSGVGKSCLLIXFSEDSF
I4TSPATNKPAAYDFLVKLLLIGDSGVGKSCLLUWS~SF
TPSFITIXGIDFKIRTIELF,GKRIKLQIUDTAGQ~TI
TPSFIATIGIDFKIRTIELEGKRIKLQIUDTAGQER.ERT1
TTAYYRGANGILLVYDVTDEKsFGNIRNWIRNIEQHATDS
Tl'AYYRGAMGILLVYDVTDEKSFGSIRNUIRNIIQHMDS
V N K N L I G N K C ~ S S R G K S I A D E Y G I K F L E T S A K
VNKIUIGNKCDMESSRGKSIADEYGIKFLETSAK
NS INVEEAFI SJAKDIKKRKIDTPNEQPQWQPGT"
NSVNVEEXFIGIAKDIKKRKIDTPND P DHTICITP
"KKKACC
N " T c c
................................... ......................................
....................................... ........................................
...................................... .......................................
....................................... ........................................
........................ .......................... . .
. . . . . . .......
Fig. 1. Comparison of the amino acid sequences of SASl and SASZ. Amino acid identities between the sequences are indicated by double dots (:). Similarities between the two sequences are based on the groupings of Dayhoff ef al. [1978] and are indicated by single dots (.).
[Gallwitz et al., 1983; Schmitt et al., 19861 and SEC4 [Salminen and Novick, 19871, which are essential for normal growth. The first -100 amino acids are -70% identical in sequence to each other with somewhat less identity extending through the remainder of the proteins. The data also show that the amino acid identities that SASl shares with YPTl and SEC4 differ. This is most apparent in the carboxy terminus. Furthermore, the SAS genes are more related to either of the yeast genes than the yeast genes are related to each other. SASl is equally similar to both YPTl and SEC4 (53.5% vs. 55.5% amino acid identity and 68.5% vs. 69.7% similarity using Dayhoff grouping [Dayhoff et al., 19781); YPTl and SEC4 are only 44.0% identical in amino acid sequence and 58.3% similar. SAS2 is 1-2% less similar than is SASl in all comparisons.
Mutations in YPTl have extreme effects on cellular properties. Cytoskeletal organization and microtubule formation are greatly disrupted [Schmitt et al., 1986; Segev and Botstein, 19871. SEC4 is one of several proteins required for protein secretion and has been directly implicated in a post-Golgi event of the pathway [Salminen and Novick, 19871. Disruption of either the YPTl or SEC4 gene inhibits normal growth; deletions yield null lethals. These data also demonstrate that neither gene can complement the other. Since the Dictyostelium genes are more related to the yeast genes it will be interesting to determine if SASl or SAS2 genes can complement the functions of YPTl or SEC4.
All four of these proteins possess two cysteine residues at the carboxy terminus. These are believed to be modified by fatty acids that anchor the proteins in the membrane. Although the ras proteins do not terminate in cysteines, they are believed to be anchored in the membrane by a fatty acid modification of a functionally equivalent cysteine residue at position 4 from the carboxy terminus (see Fig. 2).
An examination of the genomic structures of the two Dictyostelium genes
GTP-Binding Protein Genes 263
S A S l RaS R a l Rho
S A S l Ras Ral Rho
S A S l Ras Ral Rho
SASl RaS Ral Rho
SASl RaS Ral Rho
IUSPATNKSAAYDYLIKLLLI r.1 DSGV SC LIXFSEDSFl'PSFITTIGIDEKIR ~EYKLVWIGIAGGVIGI(lSAlLlTIQLIQNHFVDEMPT1EDSYRKQ
~ K G Q N S ~ I I G I S G G V I G I ( I S A I L I ~ F 1 I Y D E F V E D Y E P T K A D S Y R K K M I R K K L V I V G DGAC GK TC L LIVFSKDQFP-AD I!! I!_! I!
T I ELEGKRIloqIU r i i i G E 1 RFRTIlTAYYRGAMGILLVYDVTDEKSFGNI WIDGETCLLDIL[DTAGQE~EYSAHRIQYMRTGEGFI"TKWEDI VVLDGEEVQIDILlDTAGQEIDYAAIRDNYFRSGEGFITEKESFAAT
DYDRIRPISYPDTDVIIWCFSIDSPDSLENI
AEKKWDSSR HQYREQIKRVKDSDDVPlrVlLIVIGNKICIDIL AARTVESRQ ADFREQILRVKEDENVPFLlLlVlGNK[S[DIL EDKRQVSVEE
PEKWTPEVRHFCPNVPII L V GNK K D IRNDESTKREUKMKQEPFDG I! I!__! I! -- ----
GKSIADEYGIKFL~ E J T I SAKINSINVEEAFISIAKDIKIDTPNEWQWQ AQDIARSYGIPYI I E I TI SAKI TRQGVEDAFYTLVRF.IRQH
RAHAEKINAYSYL E C SAK TKEGVRDWETATRAAIQV
KIRK AKNRADQWNVNYV I E 1 TI SAKI TFANVDEWFFD~IRARKKEDSKIXNGKKK
KKK I! I!__!
INPPDESGPCNSCK I C I VLS
KKGG C V V L RKSIAKRIRER [ C l C I L
I! Fig. 2 Comparison of amino acid sequences of SASl, rus [human H-rus-I; Capon et ul., 19831, rul [simian; Chardin and Tavitian, 19861, and rho [Aplysiu; Madaule and Axel, 19851. The outlined regions are highly conserved areas in all members of the rus family. The cysteines at the carboxy termini are sites of palmitylation and anchor these proteins to membranes. The four other conserved regions are involved in the binding of GTP and in GTPase activity.
reveals interesting differences. SASl contains a small intron in its protein coding region, whereas SAS2 encodes an unspliced RNA. Also, the former gene encodes an mRNA whose 3'-untranslated region contains an (AAC)16 sequence.
We have also initiated a study of the expression of both genes during development of Dictyostelium discoideum. Poly (A) + RNA was purified from cells at different times during their developmental cycle and hybridized on RNA blots to a probe common to both genes (Fig. 3 ) . Two hybridizing RNA bands are detected. The smaller is present in vegetative cells and is present through the first 10 hours of development. The larger band is first detected at 10 hours of development. Its abundance level increases dramatically before again declining at very late stages of development.
It is interesting that SASl is associated with a repetitive sequence, (dAAC/dGTT),, shared by several developmentally regulated genes in Dictyostelium that appear maximally expressed during aggregate formation [Kimmel and Firtel, 1985; Saxe and Kimmel, unpublished data]. Similar sequences have been observed in some Drosophila loci involved in tissue differentiation [Wharton et al., 19851. It is
264 Saxe and Kimmel
M V 5 10 15 20
1.6 k b
Fig. 3. Developmental blot of RNA from Dicryosteliurn discoideurn. Cells were developed for up to 20 hours, poly (A)+ RNA was purified, and blots were hybridized with a 32P-labeled RNA probe corresponding to a portion of the protein-coding region of S A S l . M, size markers; V, RNA from vegetative cells.
possible that this common element plays a regulatory role for genes expressed at specific developmental stages.
By analogy with other GTP-binding proteins it is likely that these genes are required for normal growth and development, and it is possible that each gene is required for different functions. It is significant that the gene expression switch occurs in conjunction with aggregate formation and cytodifferentiation. The interactions of these proteins with their putative effector molecules may thus change during development and couple to different intracellular signalling systems.
GTP-Binding Protein Genes 265
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