Short Hydrophobic Region Research Articles

Release of a disordered domain enhances HspB1 chaperone activity toward tau

Small heat shock proteins (sHSPs) are a class of ATP-independent molecular chaperones that play vital roles in maintaining protein solubility and preventing aberrant protein aggregation. They form highly dynamic, polydisperse oligomeric ensembles and contain long intrinsically disordered regions. Experimental challenges posed by these properties have greatly impeded our understanding of sHSP structure and mechanism of action. Here we characterize interactions between the human sHSP HspB1 (Hsp27) and microtubule-associated protein tau, which is implicated in multiple dementias, including Alzheimer's disease. We show that tau binds both to a well-known binding groove within the structured alpha-crystallin domain (ACD) and to sites within the enigmatic, disordered N-terminal region (NTR) of HspB1. However, only interactions involving the NTR lead to productive chaperone activity, whereas ACD binding is uncorrelated with chaperone function. The tau-binding groove in the ACD also binds short hydrophobic regions within HspB1 itself, and HspB1 mutations that disrupt these intrinsic ACD-NTR interactions greatly enhance chaperone activity toward tau. This leads to a mechanism in which the release of the disordered NTR from a binding groove on the ACD enhances chaperone activity toward tau. The study advances understanding of the mechanisms by which sHSPs achieve their chaperone activity against amyloid-forming clients and how cells defend against pathological tau aggregation. Furthermore, the resulting mechanistic model points to ways in which sHSP chaperone activity may be increased, either by native factors within the cell or by therapeutic intervention.

Glycoprotein 3 of Porcine Reproductive and Respiratory Syndrome Virus Exhibits an Unusual Hairpin-Like Membrane Topology.

Glycoprotein 3 (GP3) of the arterivirus porcine reproductive and respiratory syndrome virus (PRRSV) consists of a cleaved signal peptide, a highly glycosylated domain, a short hydrophobic region, and an unglycosylated C-terminal domain. GP3 is supposed to form a complex with GP2 and GP4 in virus particles, but secretion of GP3 from cells has also been reported. We analyzed the membrane topology of GP3 from various PRRSV strains. A fraction of the protein is secreted from transfected cells, GP3 from PRRSV-1 strains to a greater extent than GP3 from PRRSV-2 strains. This secretion behavior is reversed after exchange of the variable C-terminal domain. A fluorescence protease protection assay shows that the C terminus of GP3, fused to green fluorescent protein (GFP), is resistant to proteolytic digestion in permeabilized cells. Furthermore, glycosylation sites inserted into the C-terminal part of GP3 are used. Both experiments indicate that the C terminus of GP3 is translocated into the lumen of the endoplasmic reticulum. Deletion of the conserved hydrophobic region greatly enhances secretion of GP3, and fusion of this domain to GFP promotes membrane anchorage. Bioinformatics suggests that the hydrophobic region forms an amphipathic helix. Accordingly, exchanging only a few amino acids in its hydrophilic face prevents secretion of GP3 and in its hydrophobic face enhances it. Exchanging the latter amino acids in the context of the viral genome did not affect release of virions, but released particles were not infectious. In sum, GP3 exhibits an unusual hairpin-like membrane topology that might explain why a fraction of the protein is secreted.IMPORTANCE PRRSV is the most important pathogen in the pork industry. It causes persistent infections that lead to reduced weight gain of piglets; highly pathogenic strains even kill 90% of an infected pig population. PRRSV cannot be eliminated from pig farms by vaccination due to the large amino acid variability between the existing strains, especially in the glycoproteins. Here, we analyzed basic structural features of GP3 from various PRRSV strains. We show that the protein exhibits an unusual hairpin-like membrane topology; membrane anchoring might occur via an amphipathic helix. This rather weak membrane anchor explains why a fraction of the protein is secreted from cells. Interestingly, PRRSV-1 strains secrete more GP3 than PRRSV-2. We speculate that secreted GP3 plays a role during PRRSV infection of pigs: it might serve as a decoy to distract antibodies away from virus particles.

Tuning crystallization pathways through sequence engineering of biomimetic polymers.

Two-step nucleation pathways in which disordered, amorphous, or dense liquid states precede the appearance of crystalline phases have been reported for a wide range of materials, but the dynamics of such pathways are poorly understood. Moreover, whether these pathways are general features of crystallizing systems or a consequence of system-specific structural details that select for direct versus two-step processes is unknown. Using atomic force microscopy to directly observe crystallization of sequence-defined polymers, we show that crystallization pathways are indeed sequence dependent. When a short hydrophobic region is added to a sequence that directly forms crystalline particles, crystallization instead follows a two-step pathway that begins with the creation of disordered clusters of 10-20 molecules and is characterized by highly non-linear crystallization kinetics in which clusters transform into ordered structures that then enter the growth phase. The results shed new light on non-classical crystallization mechanisms and have implications for the design of self-assembling polymersystems.

Two Hydrophobic Residues Can Determine the Specificity of Mitogen-activated Protein Kinase Docking Interactions

MAPKs bind to many of their upstream regulators and downstream substrates via a short docking motif (the D-site) on their binding partner. MAPKs that are in different families (e.g. ERK, JNK, and p38) can bind selectively to D-sites in their authentic substrates and regulators while discriminating against D-sites in other pathways. Here we demonstrate that the short hydrophobic region at the distal end of the D-site plays a critical role in determining the high selectivity of JNK MAPKs for docking sites in their cognate MAPK kinases. Changing just 1 or 2 key hydrophobic residues in this submotif is sufficient to turn a weak JNK-binding D-site into a strong one, or vice versa. These specificity-determining differences are also found in the D-sites of the ETS family transcription factors Elk-1 and Net. Moreover, swapping two hydrophobic residues between these D-sites switches the relative efficiency of Elk-1 and Net as substrates for ERK versus JNK, as predicted. These results provide new insights into docking specificity and suggest that this specificity can evolve rapidly by changes to just 1 or 2 amino acids.

Hierarchical Functional Specificity of Cytosolic Heat Shock Protein 70 (Hsp70) Nucleotide Exchange Factors in Yeast

Heat shock protein 70 (Hsp70) molecular chaperones play critical roles in protein homeostasis. In the budding yeast Saccharomyces cerevisiae, cytosolic Hsp70 interacts with up to three types of nucleotide exchange factors (NEFs) homologous to human counterparts: Sse1/Sse2 (Heat shock protein 110 (Hsp110)), Fes1 (HspBP1), and Snl1 (Bag-1). All three NEFs stimulate ADP release; however, it is unclear why multiple distinct families have been maintained throughout eukaryotic evolution. In this study we investigate NEF roles in Hsp70 cell biology using an isogenic combinatorial collection of NEF deletion mutants. Utilizing well characterized model substrates, we find that Sse1 participates in most Hsp70-mediated processes and is of particular importance in protein biogenesis and degradation, whereas Fes1 contributes to a minimal extent. Surprisingly, disaggregation and resolubilization of thermally denatured firefly luciferase occurred independently of NEF activity. Simultaneous deletion of SSE1 and FES1 resulted in constitutive activation of heat shock protein expression mediated by the transcription factor Hsf1, suggesting that these two factors are important for modulating stress response. Fes1 was found to interact in vivo preferentially with the Ssa family of cytosolic Hsp70 and not the co-translational Ssb homolog, consistent with the lack of cold sensitivity and protein biogenesis phenotypes for fes1Δ cells. No significant consequence could be attributed to deletion of the minor Hsp110 SSE2 or the Bag homolog SNL1. Together, these lines of investigation provide a comparative analysis of NEF function in yeast that implies Hsp110 is the principal NEF for cytosolic Hsp70, making it an ideal candidate for therapeutic intervention in human protein folding disorders.

HIV-1 Tat recruits transcription elongation factors dispersed along a flexible AFF4 scaffold

The HIV-1 Tat protein stimulates viral gene expression by recruiting human transcription elongation complexes containing P-TEFb, AFF4, ELL2, and ENL or AF9 to the viral promoter, but the molecular organization of these complexes remains unknown. To establish the overall architecture of the HIV-1 Tat elongation complex, we mapped the binding sites that mediate complex assembly in vitro and in vivo. The AFF4 protein emerges as the central scaffold that recruits other factors through direct interactions with short hydrophobic regions along its structurally disordered axis. Direct binding partners CycT1, ELL2, and ENL or AF9 act as bridging components that link this complex to two major elongation factors, P-TEFb and the PAF complex. The unique scaffolding properties of AFF4 allow dynamic and flexible assembly of multiple elongation factors and connect the components not only to each other but also to a larger network of transcriptional regulators.

Clustered hydrophobic amino acids in amphipathic helices mediate erlin1/2 complex assembly

Erlin1 and erlin2 are highly homologous, ∼40kDa, endoplasmic reticulum membrane proteins that assemble into a ring-shaped complex with a mass of ∼2MDa. How this complex is formed is not understood, but appears to involve multiple interactions, including a coiled-coil region that mediates lower-order erlin assembly, and a short hydrophobic region, termed the “assembly domain”, that mediates higher-order assembly into ∼2MDa complexes. Here we have used molecular modeling, mutagenesis and cross-linking to examine the role of the assembly domain in higher-order assembly. We find (i) that the assembly domains of erlin1 and erlin2 are amphipathic helices, (ii) that erlin1 alone and erlin2 alone can assemble into ∼2MDa complexes, (iii) that higher-order assembly is strongly inhibited by point mutations to the assembly domain, (iv) that three interacting hydrophobic residues in the assembly domain and aromaticity are essential for higher-order assembly, and (iv) that while erlins1 and 2 are equally capable of forming lower-order homo- and hetero-oligomers, hetero-oligomers are the most prevalent form when erlin1 and erlin2 are co-expressed. Overall, we conclude that the ∼2MDa erlin1/2 complex is composed of an assemblage of lower-order hetero-oligomers, probably heterotrimers, linked together by assembly domain hydrophobic residues.

SodERF3, a Novel Sugarcane Ethylene Responsive Factor (ERF), Enhances Salt and Drought Tolerance when Overexpressed in Tobacco Plants

The molecular signals and pathways that govern biotic and abiotic stress responses in sugarcane are poorly understood. Here we describe SodERF3, a sugarcane (Saccharum officinarum L. cv Ja60-5) cDNA that encodes a 201-amino acid DNA-binding protein that acts as a transcriptional regulator of the ethylene responsive factor (ERF) superfamily. Like other ERF transcription factors, the SodERF3 protein binds to the GCC box, and its deduced amino acid sequence contains an N-terminal putative nuclear localization signal (NLS). In addition, a C-terminal short hydrophobic region that is highly homologous to an ERF-associated amphiphilic repression-like motif, typical for class II ERFs, was found. Northern and Western blot analysis showed that SodERF3 is induced by ethylene. In addition, SodERF3 is induced by ABA, salt stress and wounding. Greenhouse-grown transgenic tobacco plants (Nicotiana tabacum L. cv. SR1) expressing SodERF3 were found to display increased tolerance to drought and osmotic stress.

Ligand-independent oligomerization of TLR4 regulated by a short hydrophobic region adjacent to the transmembrane domain

Toll-like receptors (TLRs) are key receptors for the activation of immune responses directed against pathogens. Among the more than 10 identified TLRs, TLR4 is the most unique because it associates with a variety of adaptor molecules for ligand recognition and signal transduction. However, the relationship between the unique characteristics and structural features of TLR4 is poorly defined. In this study, we demonstrate a novel biochemical characteristic of TLR4. TLR4, but not other TLRs, was observed as highly aggregated forms in immunoblotting. Interestingly, substitution of the transmembrane and cytoplasmic domain of TLR4 with those of other TLRs completely abolished the aggregation of TLR4. Furthermore, we found a short hydrophobic region (HR) adjacent to the transmembrane domain of TLR4; the TLR4 mutant lacking the HR was not aggregated and was nonfunctional in response to lipopolysaccharide. These results suggest that the HR may play a critical role in the functional oligomerization of TLR4.

The effect of hydrotropes on gas hydrate formation

In the research spectrum of hydrates in petroleum science and engineering, gas storage in hydrates is an area of study aimed at utilising the gas from marginal and small fields. Additives used with water can improve the gas storage capabilities of hydrates. This work discusses hydrate promotion capabilities of hydrotrope para-toluene sulfonic acid (p-TSA) when used as an additive in water for natural gas-hydrate formation. Hydrotropes are a class of compounds though amphiphilic in character, they have short hydrophobic regions and thus differ from classical surfactants, yet they display substantial ability to solubilise non-polar compounds in water [Langmuir 13 (1997) 3235]. The hydrate promotion capability of p-TSA was tested by forming hydrates with different concentration solutions in the presence of natural gas by subjecting it to hydrate formation conditions, and subsequently, after adequate formation, dissociating and measuring the gas to water ratio in hydrates. The experiments were carried out on macro scale levels to warrant more practical relevance for industrial scale hydrate formation operations. Experimental work on hydrotropes is supported with theoretical justification for the use of hydrotropes as hydrate promoters compared to micelle surfactants. The optimum p-TSA concentration and the optimum temperature–pressure conditions for hydrate formation in a batch process experiment were found as 3.5 g/l, and −10 °C at 1500 psig, respectively.

Molecular cloning and characterization of human, rat, and mouse synaptotagmin XV

Synaptotagmin (Syt) constitutes a large family of putative membrane trafficking proteins that share a short extracellular domain, a single N-terminal transmembrane domain, and C-terminal tandem C2 domains. In this study, I identified and characterized a novel member of the Syt family (named Syt XV-a) in the mouse, the rat, and humans. Although Syt XV-a protein has a short hydrophobic region at the very end of the N terminus (i.e., lacks a putative extracellular domain), biochemical and cellular analyses have indicated that the short hydrophobic region (amino acids 5–22) is sufficient for producing type I membrane topology in cultured cells, the same as in other Syt family proteins. Unlike other Syt isoforms, however, the mouse and human Syt XV have an alternative splicing isoform that lacks the C-terminal portion of the C2B domain (named Syt XV-b). Since the expression of Syt XV-a/b mRNA was mainly found in non-neuronal tissues (e.g., lung and testis) and Syt XV-a C2 domains lack Ca 2+-dependent phospholipid binding activity, Syt XV-a is classified as a non-neuronal, Ca 2+-independent Syt.

The Connecting Segment between Both Epidermal Growth Factor-like Domains in Blood Coagulation Factor IX Contributes to Stimulation by Factor VIIIa and Its Isolated A2 Domain

The light chain of activated factor IX comprises multiple interactions between both epidermal growth factor-like domains that contribute to enzymatic activity and binding of factor IXa to its cofactor factor VIIIa. To investigate the association between factor IXa-specific properties and surface-exposed structure elements, chimeras were constructed in which the interconnection between the modules Leu(84)-Thr(87) and the factor IX-specific loop Asn(89)-Lys(91) were exchanged for corresponding regions of factor X and factor VII. In absence of factor VIIIa, all chimeras displayed normal enzymatic activity. In the presence of factor VIIIa, replacement of loop Asn(89)-Lys(91) resulted in a minor reduction in factor IXa activity. However, chimeras with substitutions or insertions in the spacer between the epidermal growth factor-like domains showed a major defect in response to factor VIIIa. Of these chimeras, some displayed a normal response to isolated factor VIII A2 domain as a cofactor in factor X activation. Surprisingly, chimeras containing elongated inter-domain spacers from factor X or VII displayed reduced response to both complete factor VIIIa and the isolated A2 domain. Moreover, these chimeras still displayed effective association with immobilized A2 domain as assessed by surface plasmon resonance. We conclude that both sequence and length of the junction Leu(84)-Thr(87) between both epidermal growth factor-like domains contribute to the enhancement of factor IXa enzymatic activity that occurs upon assembly with factor VIIIa.

Identification of functionally important regions of the Saccharomyces cerevisiae mitochondrial translational activator Cbs1p

Translation of cytochrome b mRNA in yeast mitochondria requires activation by the nuclear-encoded Cbs1p. According to the current model, Cbs1p tethers cytochrome b mRNA to the inner mitochondrial membrane via interaction with the 5'-untranslated leader. Cbs1p is predicted to be a hydrophilic protein with two hydrophobic segments near the carboxyl-terminal end, which are both too short to span the membrane. Nevertheless Cbs1p is tightly associated with the mitochondrial membrane, as shown by its behaviour in extraction experiments with taurodeoxycholate. In an attempt to define functionally important regions of Cbs1p, we created a number of mutant alleles by random and directed mutagenesis. We report that a Cbs1p mutant protein lacking the mitochondrial presequence is still able to complement a Deltacbs1 strain, suggesting that the presequence does not contain essential mitochondrial targeting information. Mutations in a cluster of positively charged amino acids at the extremeC-terminus have no effect on Cbs1p function, but removal of this segment severely impairs Cbs1p function. Truncation of 12 or more amino acids from the C-terminus results in a completely defective protein. We further show that both short hydrophobic regions are essential for Cbs1p function, although membrane association is observed even in the absence of these regions.

Sequence Polymorphism in Two Novel Plasmodium vivax Ookinete Surface Proteins, Pvs25 and Pvs28, That Are Malaria Transmission-blocking Vaccine Candidates

For many malarious regions outside of Africa, development of effective transmission-blocking vaccines will require coverage against both Plasmodium falciparum and P. vivax. Work on P. vivax transmission-blocking vaccines has been hampered by the inability to clone the vaccine candidate genes from this parasite. To search for genes encoding the ookinete surface proteins from P. vivax, the DNA sequences of the eight known proteins in the P25 subfamily (Pfs25, Pgs25, Pys25, Pbs25) and in the P21/28 subfamily (Pfs28, Pgs28, Pys21, Pbs21) of zygote/ookinete surface proteins were aligned. Regions of highest identity were used to design degenerate PCR oligonucleotides. Genomic DNA from the Sal I strain of P. vivax and genomic and splinkerette DNA libraries were used as PCR templates. To characterize the polymorphisms of Pvs25 and Pvs28, these two genes were PCR amplified and the DNA sequences were determined from genomic DNA extracted from patients infected with P. vivax. Analysis of the deduced amino acid sequence of Pvs28 revealed a secretory signal sequence, four epidermal growth factor (EGF)-like domains, six copies of the heptad amino acid repeat (GSGGE/D), and a short hydrophobic region. Because the fourth EGF-like domain has four rather than six cysteines, the gene designated Pvs28 is the presumed homologue of P21/28 subfamily members. Analysis of the deduced amino acid sequence of Pvs25 revealed a similar structure to that of Pvs28. The presence of six rather than four cysteines in the fourth EGF-like domain suggested that Pvs25 is the homologue of P25 subfamily members. Several regions of genetic polymorphisms in Pvs25 and Pvs28 were identified in field isolates of P. vivax. The genes encoding two ookinete surface proteins, Pvs28 and Pvs25, from P. vivax have been isolated and sequenced. Comparison of the primary structures of Pvs25, Pvs28, Pfs25, and Pfs28 suggest that there are regions of genetic polymorphism in the P25 and P21/28 subfamilies.

Molecular Organization in Hydrotrope Assemblies

Hydrotropes are a class of compounds that are widely used in chemical, cosmetic, and pharmaceutical industries. Though amphiphilic in character, they have short hydrophobic regions and thus differ from classical surfactants. Yet they display substantial (and often selective) ability to solubilize lipophilic compounds in water. Beyond the minimal hydrotropic concentration, the molecules self-aggregate to produce the operating assembly. We show in this paper, based on crystal structure analysis of several hydrotropes, that these compounds form open-layer assemblies, reminiscent of lamellar liquid crystals consisting of alternating hydrophobic clustering of the nonpolar regions adjacent to ionic or polar regions that are knitted together in a two-dimensional network. Stacking of aromatic rings is not seen. Two types of assemblies are seen, one with a more open and extended hydrophobic layer than the other. We suggest that the solubilizates enter the hydrophobic layers of microunits producing a cooperative and mutual stabilizing effect. The observed open layer structure of hydrotropes might also account for the occasional ability of these compounds to solubilize even better than micelles.

Assignment of the human macrophage mannose receptor gene (MRC1) to 10p13 by in situ hybridization and PCR-based somatic cell hybrid mapping.

Tissue macrophages form a latticework beneath epithelial surfaces and play a pivotal role in first line host defense. The macrophage mannose receptor is highly expressed on these terminally differentiated cells, but is not expressed on circulating monocytes. Although originally defined as an endocytic receptor, the predominant physiological role for this 170-kDa transmembrane protein appears to be phagocytosis of microorganisms, such as Candida albicans and Pneumocystis carinii. The characterization of cDNAs that encode the human and mouse mannose receptors reveals that the ectodomain contains a cysteine-rich NH{sub 2} domain that appears to resemble the b subunit of ricin D. This domain is followed by a fibronectin type 2 domain. A tandem array of eight carbohydrate recognition domains bear approximately 30% homology to one another. A short hydrophobic region is followed by a 45-amino-acid cytoplasmic tail. Comparison of the human and mouse encoded proteins reveals an average homology of 80%. Of interest is the fact that the murine and human genes both span about 70 kb that contains 30 exons interrupted by 29 introns. The position of the introns and the size of the exons are identical in the mouse and human genes. 15 refs., 2 figs.

Cloning and sequence analysis of rat hepsin, a cell surface serine proteinase

A cDNA coding for the rat serine proteinase hepsin was isolated and its nucleotide sequence has been determined. The cDNA was 1739 nucleotides long and contained an open reading frame encoding a protein consisting of 416 amino-acid residues. The deduced amino-acid sequence of the rat enzyme was very similar to the human hepsin sharing an amino-acid sequence identity of 88.7%. Hydropathy plots reveal the presence of a short hydrophobic region close to the N-terminus believed to be a transmembrane domain which anchors the proteinase on the cell surface. The predicted sequence contains the His, Asp and Ser residues which make up the catalytic triad common to all serine proteinases.

Nuclear Targeting in Plants

The nucleus is the site of highly active two-way macromolecular traffic between the nucleoplasm and the cytoplasm. Nuclear import and export must be highly specific processes because the content of the nucleus is distinguished compared to the cytoplasm. This is rather amazing, considering that the nuclear envelope dissolves during mitosis of animal and plant cells, necessitating reassembly of all nuclear components and reentry of nuclear proteins into the nucleus. In addition, many basic cellular processes, such as gene transcription and cell division, clearly require proper nuclear localization of regulatory and housekeeping proteins. Furthermore, experimental evidence indicates the existence of receptors and other cellular factors that mediate nuclear transport. Nuclear transport, although extensively studied in animal and lower eukaryotic systems (3, 20), has only recently been addressed in higher plants (1, 2, 5, 7, 8, 15, 24-26). This attention to protein and nucleic acid transport into the nucleus in plants was stimulated not only by a need to understand basic cell biology, but also by the desire to modulate or regulate the expression of genes via genetic engineering. The nucleus (Fig. 1) is surrounded by the nuclear envelope, which includes the inner and outer nuclear membranes and, between them, the perinuclear space. The outer nuclear membrane is contiguous with the rough ER, and its outer surface is generally studded with ribosomes. Underlying the inner nuclear membrane is the nuclear lamina, which is composed of a network of filamentous proteins. Unlike other organelles, the nuclear envelope contains NPC2, the major sites of bidirectional protein and nucleic acid traffic. Though understood on a structural level, knowledge of the biochemistry behind the NPC is limited. Only a few proteins associated with the NPC in animal systems have been identified (3); however, their actual involvement in nuclear import has not been demonstrated. Structurally, the NPC traverses the double membrane, bringing the lipid bilayers of the inner and outer membranes together around the margins of each pore. The NPC consists of eight large protein granules arranged in a circle with a large central granule or transporter that is thought to control active nucleocytoplasmic transport of large molecules (6). In addition, eight peripheral channels of a smaller diameter would allow a passive exchange of small molecules. Therefore, transport into the nucleus is fundamentally different from that into other organelles, where transported proteins pass directly through the membrane. Although most nuclear proteins carry information that simply directs them through the nuclear pore, others must continue their journey to a final subnuclear compartment (12). With regard to what is specifically responsible for proper localization of a protein inside the nucleus, very little is known. Results obtained from yeast and animal systems have led investigators to suggest some basic rules for nuclear protein targeting (3). Proteins smaller than 40 to 60 kD are thought to diffuse through the nuclear pore, though no physiologically relevant macromolecule has been shown to traverse the nuclear pore by diffusion. However, it has been shown that many proteins require at least one NLS. Two criteria are used to define these NLSs: (a) an NLS is sufficient to redirect a cytoplasmic protein to the nucleus, and (b) an NLS is necessary for directing a nuclear protein to the nucleus. However, in many cases the NLSs are not adequately characterized by both criteria. Known NLSs can be grouped into several categories (3). First, SV40-like NLSs contain a short stretch of basic amino acids (PKKKRKV). Second, MAT a2-like NLSs consist of short hydrophobic regions that contain one or more basic amino acids (KIPIK). Third, bipartite NLSs are usually a combination of two regions of basic amino acids separated by a spacer of more than four residues (SPPKAVKRPAATKKAGQAKKKKLDKEDES) (17). The first region has two basic amino acids and the second has at least three out of five basic residues. According to current speculation, the two basic regions cooperate in binding, whereas the spacer may facilitate their cooperative interaction. In addition, there are some NLSs that do not fit any of the above-mentioned groups and that are characteristic of some viral nuclear proteins (for example, the NLS of influenza ribonucleoprotein is AAFEDLRVRS). Although NLSs have been grouped into these categories, a consensus as to sequence, either between or within groups, has not been reached. This article will focus on recent advances in our understanding of NLSs recognized in plants. For earlier reviews on nuclear targeting in animal and yeast cells, see Garcia-Bustos et al. (3) and Silver (20).

The two major membrane skeletal proteins (articulins) of Euglena gracilis define a novel class of cytoskeletal proteins.

60% of the peripheral membrane skeleton of Euglena gracilis consists of equimolar amounts of two proteins (articulins) with M(r)s in SDS gels of 80 and 86 kD. To understand eventually how these proteins assemble and function in maintaining cell form and membrane integrity we have undertaken a molecular characterization of articulins. A lambda gt11 expression library constructed from Euglena gracilis mRNAs was screened with antibodies against both articulins. Two sets of cDNAs were recovered, and evidence from three independent assays confirmed that both sets encoded articulins: (a) Anti-articulin antibodies recognized a high molecular weight beta-galactosidase (beta-gal) fusion protein expressed in bacteria infected with lambda gt11 cDNA clones. (b) Antibodies generated against the bacterially expressed beta-gal fusion protein identified one or the other articulin in Western blots of Euglena proteins. These antibodies also localized to the membrane skeletal region in thin sections of Euglena. (c) Peptide maps of the beta-gal fusion protein were similar to peptide maps of Euglena articulins. From the nucleotide sequence of the two sets of cDNAs an open reading frame for each articulin was deduced. In addition to 37% amino acid identity and overall structural similarity, both articulins exhibited a long core domain consisting of over 30 12-amino acid repeats with the consensus VPVPV--V--. Homology plots comparing the same or different articulins revealed larger, less regular repeats in the core domain that coincided with predicted turns in extended beta-sheets. Outside the core domain a short hydrophobic region containing four seven-amino acid repeats (consensus: APVTYGA) was identified near the carboxy terminus of the 80-kD articulin, but near the amino terminus of the 86-kD articulin. No extensive sequence similarities were found between articulins and other protein sequences in various databanks. We conclude that the two articulins are related members of a new class of membrane cytoskeletal proteins.

Molecular Cloning of the cDNA for Stimulatory GDP/GTP Exchange Protein for smg p21s (ras p21-Like Small GTP-Binding Proteins) and Characterization of Stimulatory GDP/GTP Exchange Protein

We have recently purified to near homogeneity the stimulatory GDP/GTP exchange protein for smg p21s (ras p21-like GTP-binding proteins) from bovine brain cytosol. This regulatory protein, named GDP dissociation stimulator (GDS), stimulates the GDP/GTP exchange reaction of smg p21s by stimulating the dissociation of GDP from and the subsequent binding of GTP to them. In this study, we have isolated and sequenced the cDNA of smg p21 GDS from a bovine brain cDNA library by using an oligonucleotide probe designed from the partial amino acid sequence of the purified smg p21 GDS. The cDNA has an open reading frame encoding a protein of 558 amino acids with a calculated Mr value of 61,066, similar to the Mr of 53,000 estimated for the purified smg p21 GDS by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and sucrose density gradient ultracentrifugation. The isolated cDNA is expressed in Escherichia coli, and the encoded protein exhibits smg p21 GDS activity. smg p21 GDS is overall hydrophilic, but there are several short hydrophobic regions. The smg p21 GDS mRNA is present in bovine brain and various rat tissues. smg p21 GDS has low amino acid sequence homology with the yeast CDC25 and SCD25 proteins, which may regulate the GDP/GTP exchange reaction of the yeast RAS2 protein, but not with ras p21 GTPase-activating protein, the inhibitory GDP/GTP exchange proteins (GDP dissociation inhibitor) for smg p25A and rho p21s, and the beta gamma subunits of heterotrimeric GTP-binding proteins such as Gs and Gi.