The Role of the Membrane-spanning Domain Sequence in Glycoprotein-mediated Membrane Fusion

The role of glycoprotein membrane-spanning domains in the process of membrane fusion is poorly understood. It has been demonstrated that replacing all or part of the membrane-spanning domain of a viral fusion protein with sequences that encode signals for glycosylphosphatidylinositol linkage attachment abrogates membrane fusion activity. It has been suggested, however, that the actual amino acid sequence of the membrane-spanning domain is not critical for the activity of viral fusion proteins. We have examined the function of Moloney murine leukemia virus envelope proteins with substitutions in the membrane-spanning domain. Envelope proteins bearing substitutions for proline 617 are processed and incorporated into virus particles normally and bind to the viral receptor. However, they possess greatly reduced or undetectable capacities for the promotion of membrane fusion and infectious virus particle formation. Our results imply a direct role for the residues in the membrane-spanning domain of the murine leukemia virus envelope protein in membrane fusion and its regulation. They also support the thesis that membrane-spanning domains possess a sequence-dependent function in other protein-mediated membrane fusion events.

Sequence Determinants for Regulated Degradation of Yeast 3-Hydroxy-3-Methylglutaryl-CoA Reductase, an Integral Endoplasmic Reticulum Membrane Protein

The degradation rate of 3-hydroxy-3-methylglutaryl CoA reductase (HMG-R), a key enzyme of the mevalonate pathway, is regulated through a feedback mechanism by the mevalonate pathway. To discover the intrinsic determinants involved in the regulated degradation of the yeast HMG-R isozyme Hmg2p, we replaced small regions of the Hmg2p transmembrane domain with the corresponding regions from the other, stable yeast HMG-R isozyme Hmg1p. When the first 26 amino acids of Hmg2p were replaced with the same region from Hmg1p, Hmg2p was stabilized. The stability of this mutant was not due to mislocalization, but rather to an inability to be recognized for degradation. When amino acid residues 27–54 of Hmg2p were replaced with those from Hmg1p, the mutant was still degraded, but its degradation rate was poorly regulated. The degradation of this mutant was still dependent on the first 26 amino acid residues and on the function of the HRD genes. These mutants showed altered ubiquitination levels that were well correlated with their degradative phenotypes. Neither determinant was sufficient to impart regulated degradation to Hmg1p. These studies provide evidence that there are sequence determinants in Hmg2p necessary for degradation and optimal regulation, and that independent processes may be involved in Hmg2p degradation and its regulation.

A Ubiquitin-Conjugating Enzyme Is Essential for Developmental Transitions in Dictyostelium

We have identified a developmentally essential gene, UbcB, by insertional mutagenesis. The encoded protein (UBC1) shows very high amino acid sequence identity to ubiquitin-conjugating enzymes from other organisms, suggesting that UBC1 is involved in protein ubiquitination and possibly degradation during Dictyostelium development. Consistent with the homology of the UBC1 protein to UBCs, the developmental pattern of protein ubiquitination is altered in ubcB-null cells. ubcB-null cells are blocked in the ability to properly execute the developmental transition that occurs between the induction of postaggregative gene expression during mound formation and the induction of cell-type differentiation and subsequent morphogenesis. ubcB-null cells plated on agar form mounds with normal kinetics; however, they remain at this stage for ∼10 h before forming multiple tips and fingers that then arrest. Under other conditions, some of the fingers form migrating slugs, but no culmination is observed. In ubcB-null cells, postaggregative gene transcripts accumulate to very high levels and do not decrease significantly with time as they do in wild-type cells. Expression of cell-type-specific genes is very delayed, with the level of prespore-specific gene expression being significantly reduced compared with that in wild-type cells. lacZ reporter studies using developmentally regulated and cell-type-specific promoters suggest that ubcB-null cells show an unusually elevated level of staining of lacZ reporters expressed in anterior-like cells, a regulatory cell population found scattered throughout the aggregate, and reduced staining of a prespore reporter. ubcB-null cells in a chimeric organism containing predominantly wild-type cells are able to undergo terminal differentiation but show altered spatial localization. In contrast, in chimeras containing only a small fraction of wild-type cells, the mature fruiting body is very small and composed almost exclusively of wild-type cells, with the ubcB-null cells being present as a mass of cells located in extreme posterior of the developing organism. The amino acid sequence analysis of the UbcB open reading frame (ORF) and the analysis of the developmental phenotypes suggest that tip formation and subsequent development requires specific protein ubiquitination, and possibly degradation.

Three-dimensional structure of human electron transfer flavoprotein to 2.1-Å resolution

Mammalian electron transfer flavoproteins (ETF) are heterodimers containing a single equivalent of flavin adenine dinucleotide (FAD). They function as electron shuttles between primary flavoprotein dehydrogenases involved in mitochondrial fatty acid and amino acid catabolism and the membrane-bound electron transfer flavoprotein ubiquinone oxidoreductase. The structure of human ETF solved to 2.1-Å resolution reveals that the ETF molecule is comprised of three distinct domains: two domains are contributed by the α subunit and the third domain is made up entirely by the β subunit. The N-terminal portion of the α subunit and the majority of the β subunit have identical polypeptide folds, in the absence of any sequence homology. FAD lies in a cleft between the two subunits, with most of the FAD molecule residing in the C-terminal portion of the α subunit. Alignment of all the known sequences for the ETF α subunits together with the putative FixB gene product shows that the residues directly involved in FAD binding are conserved. A hydrogen bond is formed between the N5 of the FAD isoalloxazine ring and the hydroxyl side chain of αT266, suggesting why the pathogenic mutation, αT266M, affects ETF activity in patients with glutaric acidemia type II. Hydrogen bonds between the 4′-hydroxyl of the ribityl chain of FAD and N1 of the isoalloxazine ring, and between αH286 and the C2-carbonyl oxygen of the isoalloxazine ring, may play a role in the stabilization of the anionic semiquinone. With the known structure of medium chain acyl-CoA dehydrogenase, we hypothesize a possible structure for docking the two proteins.

ZnT-3, a putative transporter of zinc into synaptic vesicles

The murine ZnT3 gene was cloned by virtue of its homology to the ZnT2 gene, which encodes a membrane protein that facilitates sequestration of zinc in endosomal vesicles. ZnT-3 protein is predicted to have six transmembrane domains and shares 52% amino acid identity with ZnT-2, with the homology extending throughout the two sequences. Human ZnT-3 cDNAs were also cloned; the amino acid sequence is 86% identical to murine ZnT-3. The mouse ZnT3 gene has 8 exons and maps to chromosome 5. Northern blot and reverse transcriptase–PCR analyses demonstrate that murine ZnT-3 expression is restricted to the brain and testis. In situ hybridization reveals that within the brain, ZnT-3 mRNA is most abundant in the hippocampus and cerebral cortex. Antibodies raised against the C-terminal tail of mouse ZnT-3 react with the projections from these neurons and produce a pattern similar to that obtained with Timm’s reaction, which reveals histochemically reactive zinc within synaptic vesicles. We propose that ZnT-3 facilitates the accumulation of zinc in synaptic vesicles.

Mitochondrial carbonic anhydrase CA VB: Differences in tissue distribution and pattern of evolution from those of CA VA suggest distinct physiological roles

A cDNA for a second mouse mitochondrial carbonic anhydrase (CA) called CA VB was identified by homology to the previously characterized murine CA V, now called CA VA. The full-length cDNA encodes a 317-aa precursor that contains a 33-aa classical mitochondrial leader sequence. Comparison of products expressed from cDNAs for murine CA VB and CA VA in COS cells revealed that both expressed active CAs that localized in mitochondria, and showed comparable activities in crude extracts and in mitochondria isolated from transfected COS cells. Northern blot analyses of total RNAs from mouse tissues and Western blot analyses of mouse tissue homogenates showed differences in tissue-specific expression between CA VB and CA VA. CA VB was readily detected in most tissues, while CA VA expression was limited to liver, skeletal muscle, and kidney. The human orthologue of murine CA VB was recently reported also. Comparison of the CA domain sequence of human CA VB with that reported here shows that the CA domains of CA VB are much more highly conserved between mouse and human (95% identity) than the CA domains of mouse and human CA VAs (78% identity). Analysis of phylogenetic relationships between these and other available human and mouse CA isozyme sequences revealed that mammalian CA VB evolved much more slowly than CA VA, accepting amino acid substitutions at least 4.5 times more slowly since each evolved from its respective human–mouse ancestral gene around 90 million years ago. Both the differences in tissue distribution and the much greater evolutionary constraints on CA VB sequences suggest that CA VB and CA VA have evolved to assume different physiological roles.

Interaction of 5-lipoxygenase with cellular proteins

5-Lipoxygenase (5LO) plays a pivotal role in cellular leukotriene synthesis. To identify proteins interacting with human 5LO, we used a two-hybrid approach to screen a human lung cDNA library. From a total of 1.5 × 107 yeast transformants, nine independent clones representing three different proteins were isolated and found to specifically interact with 5LO. Four 1.7- to 1.8-kb clones represented a 16-kDa protein named coactosin-like protein for its significant homology with coactosin, a protein found to be associated with actin in Dictyostelium discoideum. Coactosin-like protein thus may provide a link between 5LO and the cytoskeleton. Two other yeast clones of 1.5 kb encoded transforming growth factor (TGF) type β receptor-I-associated protein 1 partial cDNA. TGF type β receptor-I-associated protein 1 recently has been reported to associate with the activated form of the TGF β receptor I and may be involved in the TGF β-induced up-regulation of 5LO expression and activity observed in HL-60 and Mono Mac 6 cells. Finally, three identical 2.1-kb clones contained the partial cDNA of a human protein with high homology to a hypothetical helicase K12H4.8 from Caenorhabditis elegans and consequently was named ΔK12H4.8 homologue. Analysis of the predicted amino acid sequence revealed the presence of a RNase III motif and a double-stranded RNA binding domain, indicative of a protein of nuclear origin. The identification of these 5LO-interacting proteins provides additional approaches to studies of the cellular functions of 5LO.

Gβ5 prevents the RGS7-Gαo interaction through binding to a distinct Gγ-like domain found in RGS7 and other RGS proteins

The G protein β subunit Gβ5 deviates significantly from the other four members of Gβ-subunit family in amino acid sequence and subcellular localization. To detect the protein targets of Gβ5 in vivo, we have isolated a native Gβ5 protein complex from the retinal cytosolic fraction and identified the protein tightly associated with Gβ5 as the regulator of G protein signaling (RGS) protein, RGS7. Here we show that complexes of Gβ5 with RGS proteins can be formed in vitro from the recombinant proteins. The reconstituted Gβ5-RGS dimers are similar to the native retinal complex in their behavior on gel-filtration and cation-exchange chromatographies and can be immunoprecipitated with either anti-Gβ5 or anti-RGS7 antibodies. The specific Gβ5-RGS7 interaction is determined by a distinct domain in RGS that has a striking homology to Gγ subunits. Deletion of this domain prevents the RGS7-Gβ5 binding, although the interaction with Gα is retained. Substitution of the Gγ-like domain of RGS7 with a portion of Gγ1 changes its binding specificity from Gβ5 to Gβ1. The interaction of Gβ5 with RGS7 blocked the binding of RGS7 to the Gα subunit Gαo, indicating that Gβ5 is a specific RGS inhibitor.

Structural characterization of an engineered tandem repeat contrasts the importance of context and sequence in protein folding

To test a different approach to understanding the relationship between the sequence of part of a protein and its conformation in the overall folded structure, the amino acid sequence corresponding to an α-helix of T4 lysozyme was duplicated in tandem. The presence of such a sequence repeat provides the protein with “choices” during folding. The mutant protein folds with almost wild-type stability, is active, and crystallizes in two different space groups, one isomorphous with wild type and the other with two molecules in the asymmetric unit. The fold of the mutant is essentially the same in all cases, showing that the inserted segment has a well-defined structure. More than half of the inserted residues are themselves helical and extend the helix present in the wild-type protein. Participation of additional duplicated residues in this helix would have required major disruption of the parent structure. The results clearly show that the residues within the duplicated sequence tend to maintain a helical conformation even though the packing interactions with the remainder of the protein are different from those of the original helix. It supports the hypothesis that the structures of individual α-helices are determined predominantly by the nature of the amino acids within the helix, rather than the structural environment provided by the rest of the protein.

Induction of the Tat-binding protein 1 gene accompanies the disabling of oncogenic erbB receptor tyrosine kinases

Conversion of a malignant phenotype into a more normal one can be accomplished either by down-regulation of erbB family surface receptors or by creating inactive erbB heterodimers on the cell surface. In this report, we report the identification and cloning of differentially expressed genes from antibody-treated vs. untreated fibroblasts transformed by oncogenic p185neu. We repeatedly isolated a 325-bp cDNA fragment that, as determined by Northern analysis, was expressed at higher levels in anti-p185neu-treated tumor cells but not in cells expressing internalization defective p185neu receptors. This cDNA fragment was identical in amino acid sequence to the recently cloned mouse Tat binding protein-1 (mTBP1), which has 98.4% homology to the HIV tat-binding protein-1 (TBP1). TBP1 mRNA levels were found to be elevated on inhibition of the oncogenic phenotype of transformed cells expressing erbB family receptors. TBP1 overexpression diminished cell proliferation, reduced the ability of the parental cells to form colonies in vitro, and almost completely inhibited transforming efficiency in athymic mice when stably expressed in human tumor cells containing erbB family receptors. Collectively, these results suggest that the attenuation of erbB receptor signaling seems to be associated with activation/induction or recovery of a functional tumor suppressor-like gene, TBP1. Disabling erbB tyrosine kinases by antibodies or by trans-inhibition represents an initial step in triggering a TBP1 pathway.

A cytotoxic ribonuclease which specifically cleaves four isoaccepting arginine tRNAs at their anticodon loops

Colicin D has long been thought to stop protein synthesis in infected Escherichia coli cells by inactivating ribosomes, just like colicin E3. Here, we show that colicin D specifically cleaves tRNAsArg including four isoaccepting molecules both in vivo and in vitro. The cleavage occurs in vitro between positions 38 and 39 in an anticodon loop with a 2′,3′-cyclic phosphate end, and is inhibited by a specific immunity protein. Consistent with the cleavage of tRNAsArg, the RNA fraction of colicin-treated cells significantly reduced the amino acid-accepting activity only for arginine. Furthermore, we generated a single mutation of histidine in the C-terminal possible catalytic domain, which caused the loss of the killing activity in vivo together with the tRNAArg-cleaving activity both in vivo and in vitro. These findings show that colicin D directly cleaves cytoplasmic tRNAsArg, which leads to impairment of protein synthesis and cell death. Recently, we found that colicin E5 stops protein synthesis by cleaving the anticodons of specific tRNAs for Tyr, His, Asn, and Asp. Despite these apparently similar actions on tRNAs and cells, colicins D and E5 not only exhibit no sequence homology but also have different molecular mechanisms as to both substrate recognition and catalytic reaction.

Identification of two novel transmembrane γ-carboxyglutamic acid proteins expressed broadly in fetal and adult tissues

The proline-rich γ-carboxyglutamic acid (Gla) proteins (PRGPs) 1 and 2 are the founding members of a family of vitamin K-dependent single-pass integral membrane proteins characterized by an extracellular amino terminal domain of approximately 45 amino acids that is rich in Gla. The intracellular carboxyl terminal region of these two proteins contains one or two copies of the sequence PPXY, a motif present in a variety of proteins involved in such diverse cellular functions as signal transduction, cell cycle progression, and protein turnover. In this report, we describe the cloning of the cDNAs for two additional human transmembrane Gla proteins (TMG) of 20–24 kDa named TMG3 and TMG4. These two proteins possess extracellular Gla domains with 13 or 9 potential Gla residues, respectively, followed by membrane-spanning hydrophobic regions and cytoplasmic carboxyl terminal regions that contain PPXY motifs. This emerging family of integral membrane Gla proteins includes proline-rich Gla protein (PRGP) 1, PRGP2, TMG3, and TMG4, all of which are characterized by broad and variable distribution in both fetal and adult tissues. Members of this family can be grouped into two subclasses on the basis of their gene organization and amino acid sequence. These observations suggest novel physiological functions for vitamin K beyond its known role in the biosynthesis of proteins involved in blood coagulation and bone development. The identification and characterization of these proteins may allow a more complete understanding of the teratogenic consequences of exposure in utero to vitamin K antagonists, such as warfarin-based anticoagulants.

Characterization of BseMII, a new type IV restriction–modification system, which recognizes the pentanucleotide sequence 5′-CTCAG(N)10/8↓

We report the properties of the new BseMII restriction and modification enzymes from Bacillus stearothermophilus Isl 15-111, which recognize the 5′-CTCAG sequence, and the nucleotide sequence of the genes encoding them. The restriction endonuclease R.BseMII makes a staggered cut at the tenth base pair downstream of the recognition sequence on the upper strand, producing a two base 3′-protruding end. Magnesium ions and S-adenosyl-l-methionine (AdoMet) are required for cleavage. S-adenosylhomocysteine and sinefungin can replace AdoMet in the cleavage reaction. The BseMII methyltransferase modifies unique adenine residues in both strands of the target sequence 5′-CTCAG-3′/5′-CTGAG-3′. Monomeric R.BseMII in addition to endonucleolytic activity also possesses methyltransferase activity that modifies the A base only within the 5′-CTCAG strand of the target duplex. The deduced amino acid sequence of the restriction endonuclease contains conserved motifs of DNA N6-adenine methylases involved in S-adenosyl-l-methionine binding and catalysis. According to its structure and enzymatic properties, R.BseMII may be regarded as a representative of the type IV restriction endonucleases.

Gene elements that affect the longevity of rbcL sequence-containing transcripts in Chlamydomonas reinhardtii chloroplasts

The chloroplast gene rbcL encodes the large subunit of the CO2-fixing enzyme ribulose-bisphosphate carboxylase. In previous work a target for photo-accelerated degradation of Chlamydomonas reinhardtii rbcL transcripts in vivo was found to lie within the first 63 nucleotides, and a sequence element required for increasing the longevity of transcripts of rbcL-reporter genes was found to occur between nucleotides 170 and 350. Photo-accelerated degradation of rbcL transcripts has been found to require nucleotides 21 to 41. Transcript nucleotides lying between 329 and 334 and between 14 and 27 are essential for stabilizing transcripts in vivo; mutations in either region reduce the longevity of transcripts. It is postulated that the effectiveness of photo-accelerated endonuclease attacks on the nucleotide 21 to 41 region is reduced by physical blockage or distortion of the target sequence by interacting proteins that associate with nucleotides in the 14 to 27 and 329 to 334 regions of the transcripts. Both the nucleotide +329 to +334 stabilizing sequence of rbcL and a transcription enhancing sequence that lies between +126 and +170 encode well conserved (cyanobacteria through angiosperms) amino acid sequences; the evolution of expression control elements within the protein coding sequence of rbcL is considered.

Plant orthologs of p300/CBP: conservation of a core domain in metazoan p300/CBP acetyltransferase-related proteins

p300 and CBP participate as transcriptional coregulators in the execution of a wide spectrum of cellular gene expression programs controlling cell differentiation, growth and homeostasis. Both proteins act together with sequence-specific transcription factors to modify chromatin structure of target genes via their intrinsic acetyltransferase activity directed towards core histones and some transcription factors. So far, p300-related proteins have been described in animals ranging from Drosophila and Caenorhabditis elegans to humans. In this report, we describe p300/CBP-like polypeptides in the plant Arabidopsis thaliana. Interestingly, homology between animal and plant p300/CBP is largely restricted to a C-terminal segment, about 600 amino acids in length, which encompasses acetyltransferase and E1A-binding domains. We have examined whether this conservation in sequence is paralleled by a conservation in function. The same amino acid residues critical for acetyltransferase activity in human p300 are also critical for the function of one of the plant orthologs. Remarkably, plant proteins bind to the adenovirus E1A protein in a manner recapitulating the binding specificity of mammalian p300/CBP. The striking conservation of an extended segment of p300/CBP suggests that it may constitute a functional entity fulfilling functions that may be essential for all metazoan organisms.