Examining rhodopsin retention in endoplasmic reticulum and intracellular localization in vitro and in vivo by using truncated rhodopsin fragments

More than 100 mutations of rhodopsin have been identified to be associated with retinitis pigmentosa (RP), and mostly autosomal-dominant RP (ADRP). The majority of rhodopsin-associated ADRP is caused by protein misfolding and ER retention. In this study, we aimed to evaluate rhodopsin folding, exiting the ER and intracellular localization through expression of the rhodopsin fragments in COS-1 cells as well as in the transgenic zebrafish. We cloned human rhodopsin cDNA, which was then divided into the N-terminal domain, the C-terminal domain, and the fragment between the N- and C-terminal domains, and examine their intracellular expression in vitro and in vivo. We introduced a point mutation, either F45L or G51V, into this fragment and observed the intracellular localization of these mutants in COS-1 cells and in the zebrafish. The results revealed all of the truncated rhodopsin fragments except for the C-terminal domain and the full-length rhodopsin which had some plasma membrane localization, formed aggregates nearby or within the ER in COS-1 cells; however, the N-terminally truncated rhodopsin fragment, the C-terminal domain, and the full-length rhodopsin could traffic to the ROS in the zebrafish. Besides, the F45L mutation and the G51Vmutation in the rhodopsin fragment between the N- and C-terminal domains produced different effects on the aggresome formation and the intracellular distribution of the mutants both in vivo and in vitro. This current study provides new information about the mutant rhodopsin as well as in treatment of the RP in humans in the future.     

Molecular characterization of mutations of nlt-4, the pathway-specific regulatory gene which controls nitrate assimilation in Neurospora crassa

The nit-4 genes of three conventional Neurospora crassa mutations and of the closely related species, Neurospora intermedia, have been isolated by amplifying the genomic DNA with the polymerase chain reaction. Nucleotide sequencing has revealed that the three nit-4 mutants, alleles 15, 1214, and 2994, are the result of a missense mutation, a nonsense mutation and a frameshift mutation, respectively. The nucleotide sequence of the NIT4 protein coding region of a nit-4 mutant (allele 2994) and of N. intermedia have been determined and compared with that of wild-type N. crassa. The molecular characteristics confirm that the mutated gene of 2994 originated from N. intermedia and was introgressed into N. crassa. The polyglutamine domains of the N. crassa wild type, the 2994 mutant, or N. intermedia cannot replace an upstream glutamine-rich domain which is essential for nit-4 function.     

Functional characterization of missense mutations in ATP7B: Wilson disease mutation or normal variant?

Wilson disease is an autosomal recessive disorder of copper transport that causes hepatic and/or neurological disease resulting from copper accumulation in the liver and brain. The protein defective in this disorder is a putative copper-transporting P-type ATPase, ATP7B. More than 100 mutations have been identified in the ATP7B gene of patients with Wilson disease. To determine the effect of Wilson disease missense mutations on ATP7B function, we have developed a yeast complementation assay based on the ability of ATP7B to complement the high-affinity iron-uptake deficiency of the yeast mutant ccc2. We characterized missense mutations found in the predicted membrane-spanning segments of ATP7B. Ten mutations have been made in the ATP7B cDNA by site-directed mutagenesis: five Wilson disease missense mutations, two mutations originally classified as possible disease-causing mutations, two putative ATP7B normal variants, and mutation of the cysteine-proline-cysteine (CPC) motif conserved in heavy-metal-transporting P-type ATPases. All seven putative Wilson disease mutants tested were able to at least partially complement ccc2 mutant yeast, indicating that they retain some ability to transport copper. One mutation was a temperature-sensitive mutation that was able to complement ccc2 mutant yeast at 30 degreesC but was unable to complement at 37 degreesC. Mutation of the CPC motif resulted in a nonfunctional protein, which demonstrates that this motif is essential for copper transport by ATP7B. Of the two putative ATP7B normal variants tested, one resulted in a nonfunctional protein, which suggests that it is a disease-causing mutation.     

Structural implications of mutations in the pea SYM8 symbiosis gene, the DMI1 ortholog, encoding a predicted ion channel

The Pisum sativum SYM8 gene plays an essential part in both rhizobial and mycorrhizal symbioses. Mutation of sym8 in the original type line R25 blocks nodulation, mycorrhization, and Nod-factor-induced calcium spiking, an early component of the nodulation signaling pathway. We describe four new sym8 alleles of pea, which fall into the same complementation group as R25. The sym8 mutants are phenotypically similar to Medicago truncatula dmi1 mutants and map to a syntenic location. We used sequence homology to isolate the pea ortholog of M. truncatula DMI1 and have shown that the cloned pea ortholog can complement a M. truncatula dmi1 mutant for nodulation. Each of the five pea sym8 mutants carries a mutation in the DMI1 ortholog, confirming that the pea SYM8 is the DMI1 ortholog. Based on predicted structural similarities with an archaebacterial ion channel, we propose that SYM8 forms a tetrameric calcium-gated channel of a predicted structure similar to the archaebacterial potassium channel but containing a filter region that is different. The predicted structure identifies four aspartate residues (one from each subunit) forming the channel opening. We made a mutation changing the aspartate to valine and identified a missense mutation (changing alanine to valine adjacent to the aspartate residues) in this predicted filter region; both mutations caused a loss of function. We also identified a loss-of-function missense mutation (changing arginine to isoleucine) in a domain proposed to link the predicted channel and the gating ring domains, indicating that this mutation may block function by preventing a protein conformational change being transmitted from the gating-ring domain to the pore domain.     

Mutations in the extracellular domain and in the membrane-spanning domains interfere with nicotinic acetylcholine receptor maturation

The deg-3(u662) mutation is a degeneration-causing mutation in a Caenorhabditis elegans nicotinic acetylcholine receptor. In a large screen for mutations that suppress the deleterious effects of this mutation we identified 32 mutations in the deg-3 gene. Among these, 11 are missense mutations, affecting seven residues within the extracellular domain or the membrane-spanning domains. All of these mutations greatly reduce the degeneration-causing activity of deg-3(u662). All but one of these mutations cause defective localization of the DEG-3 protein, as seen in immunohistochemical analysis. Thus our screen identifies multiple residues within the nicotinic acetylcholine receptor needed for normal folding, assembly, or trafficking of this receptor. Interestingly, these mutations lead to distinct localization defects suggesting differences in their effect on DEG-3's maturation process. Specifically, mutations in the extracellular domain lead to a phenotype more severe than mutations in the membrane-spanning domains. Differences in the effects of the mutations are also predicted by homology-based modeling, showing that some mutations in the extracellular domain are likely to disrupt the native fold of the protein, while others are likely to disrupt trafficking.     

Mapping the functional topology of the animal fatty acid synthase by mutant complementation in vitro

An in vitro mutant complementation approach has been used to map the functional topology of the animal fatty acid synthase. A series of knockout mutants was engineered, each mutant compromised in one of the seven functional domains, and heterodimers generated by hybridizing all possible combinations of the mutated subunits were isolated and characterized. Heterodimers comprised of a subunit containing either a beta-ketoacyl synthase or malonyl/acetyltransferase mutant, paired with a subunit containing mutations in any one of the other five domains, are active in fatty acid synthesis. Heterodimers in which both subunits carry a knockout mutation in either the dehydrase, enoyl reductase, keto reductase, or acyl carrier protein are inactive. Heterodimers comprised of a subunit containing a thioesterase mutation paired with a subunit containing a mutation in either the dehydrase, enoyl reductase, beta-ketoacyl reductase, or acyl carrier protein domains exhibit very low fatty acid synthetic ability. The results are consistent with a model for the fatty acid synthase in which the substrate loading and condensation reactions are catalyzed by cooperation of an acyl carrier protein domain of one subunit with the malonyl/acetyltransferase or beta-ketoacyl synthase domains, respectively, of either subunit. The beta-carbon-processing reactions, responsible for the complete reduction of the beta-ketoacyl moiety following each condensation step, are catalyzed by cooperation of an acyl carrier protein domain with the beta-ketoacyl reductase, dehydrase, and enoyl reductase domains associated exclusively with the same subunit. The chain-terminating reaction is carried out most efficiently by cooperation of an acyl carrier protein domain with the thioesterase domain of the same subunit. These results are discussed in the context of a revised model for the fatty acid synthase.     

Opsin stability and folding: the role of Cys185 and abnormal disulfide bond formation in the intradiscal domain

The structure in the extracellular, intradiscal domain of rhodopsin surrounding the Cys110–Cys187 disulfide bond has been shown to be important for correct folding of this receptor in vivo. Retinitis pigmentosa misfolding mutants of the apoprotein opsin (such as P23H) misfold, as defined by a deficiency in ability to bind 11-cis retinal and form rhodopsin. These mutants also possess an abnormal Cys185–Cys187 disulfide bond in the intradiscal domain. Here, by mutating Cys185 to alanine, we eliminate the possibility of forming this abnormal disulfide bond and investigate the effect of combining the C185A mutation with the retinitis pigmentosa mutation P23H. Both the P23H and P23H/C185A double mutant suffer from low expression and poor 11-cis retinal binding. Our data suggest that misfolding events occur that do not have an absolute requirement for abnormal Cys185–Cys187 disulfide bond formation. In the detergent-solubilised, purified state, the C185A mutation allows formation of rhodopsin at wild-type (WT) levels, but has interesting effects on protein stability. C185A rhodopsin is less thermally stable than WT, whereas C185A opsin shows the same ability to regenerate rhodopsin in detergent as WT. Purified C185A and WT opsins, however, have contrasting 11-cis retinal binding kinetics. A high proportion of C185A opsin binds 11-cis retinal with a slow rate that reflects a denatured state of opsin reverting to a fast-binding, open-pocket conformation. This slower rate is not observed in a stabilising lipid/detergent system, 1,2-dimyristoyl-sn-glycero-3-phosphocholine/Chaps, in which C185A exhibits WT (fast) retinal binding. We propose that the C185A mutation destabilises the open-pocket conformation of opsin in detergent resulting in an equilibrium between correctly folded and denatured states of the protein. This equilibrium can be driven towards the correctly folded rhodopsin state by the binding of 11-cis retinal.     

Requirements for translocation of periplasmic domains in polytopic membrane proteins.

Periplasmic domains of cytoplasmic membrane proteins require export signals for proper translocation. These signals were studied by using a MalF-alkaline phosphatase fusion in a genetic selection that allowed the isolation of mislocalization mutants. In the original construct, alkaline phosphatase is fused to the second periplasmic domain of the membrane protein, and its activity is thus confined exclusively to the periplasm. Mutants that no longer translocated alkaline phosphatase were selected by complementation of a serB mutation. A total of 11 deletions in the amino terminus were isolated, all of which spanned at least the third transmembrane segment. This domain immediately precedes the periplasmic domain to which alkaline phosphatase was fused. Our results obtained in vivo support the model that amino-terminal membrane-spanning segments are required for translocation of large periplasmic domains. In addition, we found that the inability to export the alkaline phosphatase domain could be suppressed by a mutation, prlA4, in the secretion apparatus.     

Isolation and characterization of temperature-sensitive and thermostable mutants of the human receptor-like protein tyrosine phosphatase LAR.

Human LAR is a transmembrane receptor-like protein whose cytoplasmic region contains two tandemly duplicated domains homologous to protein tyrosine phosphatases (PTPases). Whereas the membrane-proximal domain I has enzymatic activity, the membrane-distal domain II has no apparent catalytic activity but seems to have a regulatory function. In order to study structure-function relationships of the LAR PTPase, LAR domain I was expressed in Escherichia coli, and mutants that have reduced catalytic activity or reduced thermostability were isolated and characterized. We isolated 18 unique hydroxylamine-induced missense mutations in the LAR domain I segment, of which three were temperature-sensitive. Five additional temperature-sensitive mutations were isolated using N-methyl-N'-nitro-N-nitrosoguanidine. All eight temperature-sensitive mutations are confined within a short segment of the LAR domain I sequence between amino acid positions 1329 and 1407. To examine whether this region is particularly prone to temperature-sensitive mutations, tyrosine at amino acid position 1379 was changed to a phenylalanine by oligonucleotide-directed mutagenesis. This mutant, Y1379-F, was indeed temperature-sensitive. We also isolated a revertant of a temperature-sensitive mutant. The revertant contained a second-site mutation (C1446-Y) that suppresses several temperature-sensitive mutations and also enhances the folding of LAR protein produced in E. coli.     

Residues in the N-Terminal Domain of MutL Required for Mismatch Repair in Bacillus subtilis

Mismatch repair is a highly conserved pathway responsible for correcting DNA polymerase errors incorporated during genome replication. MutL is a mismatch repair protein known to coordinate several steps in repair that ultimately results in strand removal following mismatch identification by MutS. MutL homologs from bacteria to humans contain well-conserved N-terminal and C-terminal domains. To understand the contribution of the MutL N-terminal domain to mismatch repair, we analyzed 14 different missense mutations in Bacillus subtilis MutL that were conserved with missense mutations identified in the human MutL homolog MLH1 from patients with hereditary nonpolyposis colorectal cancer (HNPCC). We characterized missense mutations in or near motifs important for ATP binding, ATPase activity, and DNA binding. We found that 13 of the 14 missense mutations conferred a substantial defect to mismatch repair in vivo, while three mutant alleles showed a dominant negative increase in mutation frequency to wild-type mutL. We performed immunoblot analysis to determine the relative stability of each mutant protein in vivo and found that, although most accumulated, several mutant proteins failed to maintain wild-type levels, suggesting defects in protein stability. The remaining missense mutations located in areas of the protein important for DNA binding, ATP binding, and ATPase activities of MutL compromised repair in vivo. Our results define functional residues in the N-terminal domain of B. subtilis MutL that are critical for mismatch repair in vivo.     

A Golgi retention signal in a membrane-spanning domain of coronavirus E1 protein

The E1 glycoprotein from an avian coronavirus is a model protein for studying retention in the Golgi complex. In animal cells expressing the protein from cDNA, the E1 protein is targeted to cis Golgi cisternae (Machamer, C. E., S. A. Mentone, J. K. Rose, and M. G. Farquhar. 1990. Proc. Natl. Acad. Sci. USA. 87:6944-6948). We show that the first of the three membrane-spanning domains of the E1 protein can retain two different plasma membrane proteins in the Golgi region of transfected cells. Both the vesicular stomatitis virus G protein and the alpha-subunit of human chorionic gonadotropin (anchored to the membrane by fusion with the G protein membrane-spanning domain and cytoplasmic tail) were retained in the Golgi region of transfected cells when their single membrane-spanning domains were replaced with the first membrane-spanning domain from E1. Single amino acid substitutions in this sequence released retention of the chimeric G protein, as well as a mutant E1 protein which lacks the second and third membrane-spanning domains. The important feature of the retention sequence appears to be the uncharged polar residues which line one face of a predicted alpha helix. This is the first retention signal to be defined for a resident Golgi protein. The fact that it is present in a membrane-spanning domain suggests a novel mechanism of retention in which the membrane composition of the Golgi complex plays an instrumental role in retaining its resident proteins.     

Domain structure of 3-hydroxy-3-methylglutaryl coenzyme A reductase, a glycoprotein of the endoplasmic reticulum

We present and evaluate a model for the secondary structure and membrane orientation of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the glycoprotein of the endoplasmic reticulum that controls the rate of cholesterol biosynthesis. This model is derived from proteolysis experiments that separate the 97-kilodalton enzyme into two domains, an NH2-terminal membrane-bound domain of 339 residues and a COOH-terminal water-soluble domain of 548 residues that projects into the cytoplasm and contains the catalytic site. These domains were identified by reaction with antibodies against synthetic peptides corresponding to specific regions in the molecule. Computer modeling of the reductase structure, based on the amino acid sequence as determined by molecular cloning, predicts that the NH2-terminal domain contains 7 membrane-spanning regions. Analysis of the gene structure reveals that each proposed membrane-spanning region is encoded in a separate exon and is separated from the adjacent membrane-spanning region by an intron. The COOH-terminal domain of the reductase is predicted to contain two beta-structures flanked by a series of amphipathic helices, which together may constitute the active site. The NH2-terminal membrane-bound domain of the reductase bears some resemblance to rhodopsin, the photoreceptor protein of retinal rod disks and the only other intracellular glycoprotein whose amino acid sequence is known.     

Missense substitutions lethal to essential functions of EF-Tu

We have used a simple selection and screening method to isolate function defective mutants of EF-Tu. From 28 mutants tested, 12 different missense substitutions, individually lethal to some essential function of EF-Tu, were identified by sequencing. In addition we found a new non-lethal missense mutation. The frequency of isolation of unique mutations suggests that this method can be used to easily isolate many more. The lethal mutations occur in all three structural domains of EF-Tu, but most are in domain II. We aim to use these mutants to define functional domains on EF-Tu.     

A specific transmembrane domain of a coronavirus E1 glycoprotein is required for its retention in the Golgi region

The E1 glycoprotein of the avian coronavirus infectious bronchitis virus contains a short, glycosylated amino-terminal domain, three membrane-spanning domains, and a long carboxy-terminal cytoplasmic domain. We show that E1 expressed from cDNA is targeted to the Golgi region, as it is in infected cells. E1 proteins with precise deletions of the first and second or the second and third membrane-spanning domains were glycosylated, thus suggesting that either the first or third transmembrane domain can function as an internal signal sequence. The mutant protein with only the first transmembrane domain accumulated intracellularly like the wild-type protein, but the mutant protein with only the third transmembrane domain was transported to the cell surface. This result suggests that information specifying accumulation in the Golgi region resides in the first transmembrane domain, and provides the first example of an intracellular membrane protein that is transported to the plasma membrane after deletion of a specific domain.     

LMP-1's transmembrane domains encode multiple functions required for LMP-1's efficient signaling

The latent membrane protein-1 (LMP-1) of Epstein-Barr virus (EBV) contributes to the proliferation of infected B lymphocytes by signaling through its binding to cellular signaling molecules. It apparently mimics members of the tumor necrosis factor receptor family, in particular, CD40, by binding a similar set of cellular molecules as does CD40. LMP-1 differs dramatically in its structure from CD40. LMP-1 has six membrane-spanning domains as opposed to CD40's one. LMP-1 also differs from CD40 in its apparent independence of a ligand for its signaling. We have examined the role of LMP-1's membrane-spanning domains in its signaling. Their substitution with six membrane-spanning domains from the LMP-2A protein of EBV yields a derivative which neither coimmunoprecipitates with LMP-1 nor signals to increase the activity of NF-kappaB as does wild-type LMP-1. These observations indicate that LMP-1 has specific sequences in its membrane-spanning domains required for these activities. LMP-1's first and sixth membrane-spanning domains have multiple leucine residues potentially similar to leucine-heptad motifs that can mediate protein-protein interactions in membranes (Gurezka et al., J. Biol. Chem. 274:9265-9270, 1999). Substitution of seven leucines in LMP-1's sixth membrane-spanning domain has no effect on its function, whereas similar substitutions in its first membrane-spanning domain yielded a derivative which aggregates as does wild-type LMP-1 but has only 3% of wild-type's ability to signal through NF-kappaB. Importantly, this derivative complements a mutant of LMP-1 with wild-type membrane-spanning domains but no carboxy-terminal signaling domain. These findings together indicate that the membrane-spanning domains of LMP-1 contribute multiple functions to its signaling.     

spe-10 encodes a DHHC-CRD zinc-finger membrane protein required for endoplasmic reticulum/Golgi membrane morphogenesis during Caenorhabditis elegans spermatogenesis

C. elegans spermatogenesis employs lysosome-related fibrous body-membranous organelles (FB-MOs) for transport of many cellular components. Previous work showed that spe-10 mutants contain FB-MOs that prematurely disassemble, resulting in defective transport of FB components into developing spermatids. Consequently, spe-10 spermatids are smaller than wild type and contain defective FB-MO derivatives. In this article, we show that spe-10 encodes a four-pass integral membrane protein that has a DHHC-CRD zinc-finger motif. The DHHC-CRD motif is found in a large, diverse family of proteins that have been implicated in palmitoyl transfer during protein lipidation. Seven spe-10 mutants were analyzed, including missense, nonsense, and deletion mutants. An antiserum to SPE-10 showed significant colocalization with a known marker for the FB-MOs during wild-type spermatogenesis. In contrast, the spe-10(ok1149) deletion mutant lacked detectable SPE-10 staining; this mutant lacks a spe-10 promoter and most coding sequence. The spe-10(eb64) missense mutation, which changes a conserved residue within the DHHC-CRD domain in all homologues, behaves as a null mutant. These results suggest that wild-type SPE-10 is required for the MO to properly deliver the FB to the C. elegans spermatid and the DHHC-CRD domain is essential for this function.     

The HXT1 gene product of Saccharomyces cerevisiae is a new member of the family of hexose transporters.

Two novel genes affecting hexose transport in the yeast Saccharomyces cerevisiae have been identified. The gene HXT1 (hexose transport), isolated from plasmid pSC7, was sequenced and found to encode a hydrophobic protein which is highly homologous to the large family of sugar transporter proteins from eucaryotes and procaryotes. Multicopy expression of the HXT1 gene restored high-affinity glucose transport to the snf3 mutant, which is deficient in a significant proportion of high-affinity glucose transport. HXT1 was unable to complement the snf3 growth defect in low copy number. The HXT1 protein was found to contain 12 putative membrane-spanning domains with a central hydrophilic domain and hydrophilic N- and C-terminal domains. The HXT1 protein is 69% identical to GAL2 and 66% identical to HXT2, and all three proteins were found to have a putative leucine zipper motif at a consensus location in membrane-spanning domain 2. Disruption of the HXT1 gene resulted in loss of a portion of high-affinity glucose and mannose transport, and wild-type levels of transport required both the HXT1 and SNF3 genes. Unexpectedly, expression of beta-galactosidase activity by using a fusion of the lacZ gene to the HXT1 promoter in a multicopy plasmid was maximal during lag and early exponential phases of growth, decreasing approximately 100-fold upon further entry into exponential growth. Deletion analysis of pSC7 revealed the presence of another gene (called ORF2) capable of suppressing the snf3 null mutant phenotype by restoring high-affinity glucose transport and increased low-affinity transport.     

The Lebanese allele at the low density lipoprotein receptor locus. Nonsense mutation produces truncated receptor that is retained in endoplasmic reticulum

We here describe a mutant low density lipoprotein receptor gene that produces a shortened receptor protein lacking three domains: the region of clustered O-linked carbohydrates, the membrane-spanning region, and the cytoplasmic tail. The defect is attributable to a single nucleotide substitution that creates a premature termination codon at amino acid 660, eliminating 180 residues from the mature protein. The truncated protein retains only two domains: a complete ligand-binding region (residues 1-292) and a partial epidermal growth factor precursor homology region (residues 293-659). The termination codon occurs in the middle of a cysteine-rich sequence that is part of the epidermal growth factor precursor homology domain. The mutant protein is present in markedly reduced amounts and may be translated at a reduced rate. After synthesis, most of the receptor remains within the cell for several hours with its N-linked carbohydrate in an unprocessed endoglycosidase H-sensitive form. This finding suggests that the shortened receptor leaves the endoplasmic reticulum at an abnormally slow rate, which is likely attributable to abnormal folding of the truncated protein. The mutation creates a new restriction site for the enzyme HinfI, thus permitting diagnosis by Southern blotting of genomic DNA. Two copies of this mutant gene were present in each of four unrelated Arab patients with homozygous familial hypercholesterolemia (three from Lebanon and one from Syria). We believe that this mutation, hereafter referred to as the "Lebanese allele," is responsible for the extraordinarily high incidence of familial hypercholesterolemia in Lebanon.     

Side chain and backbone contributions of Phe508 to CFTR folding

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), an integral membrane protein, cause cystic fibrosis (CF). The most common CF-causing mutant, deletion of Phe508, fails to properly fold. To elucidate the role Phe508 plays in the folding of CFTR, missense mutations at this position were generated. Only one missense mutation had a pronounced effect on the stability and folding of the isolated domain in vitro. In contrast, many substitutions, including those of charged and bulky residues, disrupted folding of full-length CFTR in cells. Structures of two mutant nucleotide-binding domains (NBDs) reveal only local alterations of the surface near position 508. These results suggest that the peptide backbone plays a role in the proper folding of the domain, whereas the side chain plays a role in defining a surface of NBD1 that potentially interacts with other domains during the maturation of intact CFTR.     

The new gene mukB codes for a 177 kd protein with coiled-coil domains involved in chromosome partitioning of E. coli.

An Escherichia coli temperature sensitive mutant which produces spontaneously normal size anucleate cells at low temperature was isolated. The mutant is defective in a previously undescribed gene, named mukB, located at 21 min on the chromosome. The mukB gene codes for a large protein (approximately 180 kd). A 1534 amino acid protein (176,826 daltons) was deduced from the nucleotide sequence of the mukB gene. Computer analysis revealed that the predicted MukB protein has distinct domains: an amino-terminal globular domain containing a nucleotide binding sequence, a central region containing two alpha-helical coiled-coil domains and one globular domain, and a carboxyl-terminal globular domain which is rich in Cys, Arg and Lys. A 180 kd protein detected in wild-type cell extracts by electrophoresis is absent in mukB null mutants. Although the null mutants are not lethal at low temperature, the absence of MukB leads to aberrant chromosome partitioning. At high temperature the mukB null mutants cannot form colonies and many nucleoids are distributed irregularly along elongated cells. We conclude that the MukB protein is required for chromosome partitioning in E. coli.