Signal and membrane anchor functions overlap in the type II membrane protein I gamma CAT

I gamma CAT is a hybrid protein that inserts into the membrane of the endoplasmic reticulum as a type II membrane protein. These proteins span the membrane once and expose the NH2-terminal end on the cytoplasmic side and the COOH terminus on the exoplasmic side. I gamma CAT has a single hydrophobic segment of 30 amino acid residues that functions as a signal for membrane insertion and anchoring. The signal-anchor region in I gamma CAT was analyzed by deletion mutagenesis from its COOH-terminal end (delta C mutants). The results show that the 13 amino acid residues on the amino-terminal side of the hydrophobic segment are not sufficient for membrane insertion and translocation. Mutant proteins with at least 16 of the hydrophobic residues are inserted into the membrane, glycosylated, and partially proteolytically processed by a microsomal protease (signal peptidase). The degree of processing varies between different delta C mutants. Mutant proteins retaining 20 or more of the hydrophobic amino acid residues can span the membrane like the parent I gamma CAT protein and are not proteolytically processed. Our data suggest that in the type II membrane protein I gamma CAT, the signals for membrane insertion and anchoring are overlapping and that hydrophilic amino acid residues at the COOH-terminal end of the hydrophobic segment can influence cleavage by signal peptidase. From this and previous work, we conclude that the function of the signal-anchor sequence in I gamma CAT is determined by three segments: a positively charged NH2 terminus, a hydrophobic core of at least 16 amino acid residues, and the COOH-terminal flanking hydrophilic segment.     

The solution structure of bacteriophage lambda protein W, a small morphogenetic protein possessing a novel fold

Protein W (gpW) from bacteriophage lambda is required for the stabilization of DNA within the phage head and for attachment of tails onto the head during morphogenesis. Although comprised of only 68 residues, it likely interacts with at least two other proteins in the mature phage and with DNA. Thus, gpW is an intriguing subject for detailed structural studies. We have determined its solution structure using NMR spectroscopy and have found it to possesses a novel fold consisting of two alpha-helices and a single two-stranded beta-sheet arranged around a well-packed hydrophobic core. The 14 C-terminal residues of gpW, which are essential for function, are unstructured in solution.     

Role of S'1 loop residues in the substrate specificities of pepsin A and chymosin

Proteolytic specificities of human pepsin A and monkey chymosin were investigated with a variety of oligopeptides as substrates. Human pepsin A had a strict preference for hydrophobic/aromatic residues at P'1, while monkey chymosin showed a diversified preferences accommodating charged residues as well as hydrophobic/aromatic ones. A comparison of residues forming the S'1 subsite between mammalian pepsins A and chymosins demonstrated the presence of conservative residues including Tyr(189), Ile(213), and Ile(300) and group-specific residues in the 289-299 loop region near the C terminus. The group-specific residues consisted of hydrophobic residues in pepsin A (Met(289), Leu/Ile/Val(291), and Leu(298)) and charged or polar residues in chymosins (Asp/Glu(289) and Gln/His/Lys(298)). Because the residues in the loop appeared to be involved in the unique specificities of respective types of enzymes, site-directed mutagenesis was undertaken to replace pepsin-A-specific residues by chymosin-specific ones and vice versa. A yeast expression vector for glutathione-S-transferase fusion protein was newly developed for expression of mutant proteins. The specificities of pepsin-A mutants could be successfully altered to the chymosin-like preference and those of chymosin mutants, to pepsin-like specificities, confirming residues in the S'1 loop to be essential for unique proteolytic properties of the enzymes. An increase in preference for charged residues at P'1 in pepsin-A mutants might have been due to an increase in the hydrogen-bonding interactions. In chymosin mutants, the reverse is possible. The changes in the catalytic efficiency for peptides having charged residues at P'1 were dominated by k(cat) rather than K(m) values.     

Two exposed amino acid residues confer thermostability on a cold shock protein

Thermophilic organisms produce proteins of exceptional stability. To understand protein thermostability at the molecular level we studied a pair of cold shock proteins, one of mesophilic and one of thermophilic origin, by systematic mutagenesis. Although the two proteins differ in sequence at 12 positions, two surface-exposed residues are responsible for the increase in stability of the thermophilic protein (by 15.8 kJ mol-1 at 70 degrees C). 11.5 kJ mol-1 originate from a predominantly electrostatic contribution of Arg 3 and 5.2 kJ mol-1 from hydrophobic interactions of Leu 66 at the carboxy terminus. The mesophilic protein could be converted to a highly thermostable form by changing the Glu residues at positions 3 and 66 to Arg and Leu, respectively. The variation of surface residues may thus provide a simple and powerful approach for increasing the thermostability of a protein.     

The elastin-binding protein of Staphylococcus aureus (EbpS) is expressed at the cell surface as an integral membrane protein and not as a cell wall-associated protein.

The elastin-binding proteins EbpS of Staphylococcus aureus strains Cowan and 8325-4 were predicted from sequence analysis to comprise 486 residues. Specific antibodies were raised against an N-terminal domain (residues 1-267) and a C-terminal domain (residues 343-486) expressed as recombinant proteins in Escherichia coli. Western blotting of lysates of wild-type 8325-4 and Newman and the corresponding ebpS mutants showed that EbpS migrated with an apparent molecular mass of 83 kDa. The protein was found exclusively in cytoplasmic membrane fractions purified from protoplasts or lysed cells, in contrast to the clumping factor ClfA, which was cell-wall-associated. EbpS was predicted to have three hydrophobic domains H1-(205-224), H2-(265-280), and H3-(315-342). A series of hybrid proteins was formed between EbpS at the N terminus and either alkaline phosphatase or beta-galactosidase at the C terminus (EbpS-PhoA, EbpS-LacZ). PhoA and LacZ were fused to EbpS between hydrophobic domains H1-H2 and H2-H3, and distal to H3. Expression of enzymatic activity in E. coli showed that EbpS is an integral membrane protein with two membrane-spanning domains H1 and H3. N-terminal residues 1-205 and C-terminal residues 343-486 were predicted to be exposed on the outer face of the cytoplasmic membrane. The ligand-binding domain of EbpS is known from previous studies to be present in the N terminus between residues 14-34 and probing whole cells with anti-EbpS1-267 antibodies indicated that this region is exposed on the surface of intact cells. This was also confirmed by the observation that wild-type S. aureus Newman cells bound labeled tropoelastin whereas the ebpS mutant bound 72% less. In contrast, the C terminus, which carries a putative LysM peptidoglycan-binding domain, is not exposed on the surface of intact cells and presumably remains buried within the peptidoglycan. Finally, expression of EbpS was correlated with the ability of cells to grow to a higher density in liquid culture, suggesting that EbpS may have a role in regulating cell growth.     

COOH-terminal requirements for the correct processing of a phosphatidylinositol-glycan anchored membrane protein

Placental alkaline phosphatase (PLAP) is anchored to the plasma membrane by a phosphatidylinositol-glycan (PI-G) moiety. During processing of nascent PLAP, a 29-residue COOH-terminal peptide is cleaved out and the PI-G moiety is attached to the newly created COOH terminus of the mature protein. To investigate the structural requirements of the COOH terminus of the nascent protein for PI-G tailing and anchoring to the plasma membrane, we have transfected COS cells with wild type and mutant forms of cDNA encoding human prepro-PLAP. Utilizing a series of COOH-terminal deletion mutants of prepro-PLAP, it was found that to be PI-G-tailed the newly synthesized protein must possess an uncharged, predominantly hydrophobic amino acid sequence of a minimal length in the COOH-terminal peptide. While forms of prepro-PLAP with 17 consecutive hydrophobic residues in the terminal sequence yielded PI-G-tailed and membrane-bound products, prepro-PLAP mutants with 13 or fewer of such residues yielded hydrophilic proteins that were no longer PI-G-tailed but efficiently secreted into the medium. Studies using cassette mutants demonstrated that the precise amino sequence of the COOH-terminal region could be altered as long as minimal hydrophobicity and length was maintained.     

Mutation of single hydrophobic residue I27, L35, F39, L58, L65, L67, or L71 in the N terminus of VP5 abolishes interaction with the scaffold protein and prevents closure of herpes simplex virus type 1 capsid shells

Protein-protein interactions drive the assembly of the herpes simplex virus type 1 (HSV-1) capsid. A key interaction occurs between the C-terminal tail of the scaffold protein (pre-22a) and the major capsid protein (VP5). Previously (Z. Hong, M. Beaudet-Miller, J. Durkin, R. Zhang, and A. D. Kwong, J. Virol. 70:533-540, 1996) it was shown that the minimal domain in the scaffold protein necessary for this interaction was composed of a hydrophobic amphipathic helix. The goal of this study was to identify the hydrophobic residues in VP5 important for this bimolecular interaction. Results from the genetic analysis of second-site revertant virus mutants identified the importance of the N terminus of VP5 for the interaction with the scaffold protein. This allowed us to focus our efforts on a small region of this large polypeptide. Twenty-four hydrophobic residues, starting at L23 and ending at F84, were mutated to alanine. All the mutants were first screened for interaction with pre-22a in the yeast two-hybrid assay. From this in vitro assay, seven residues, I27, L35, F39, L58, L65, L67, and L71, that eliminated the interaction when mutated were identified. All 24 mutants were introduced into the virus genome with a genetic marker rescue/marker transfer system. For this system, viruses and cell lines that greatly facilitated the introduction of the mutants into the genome were made. The same seven mutants that abolished interaction of VP5 with pre-22a resulted in an absolute requirement for wild-type VP5 for growth of the viruses. The viruses encoding these mutations in VP5 were capable of forming capsid shells comprised of VP5, VP19C, VP23, and VP26, but the closure of these shells into an icosahedral structure was prevented. Mutation at L75 did not affect the ability of this protein to interact with pre-22a, as judged from the in vitro assay, but this mutation specified a lethal effect for virus growth and abolished the formation of any detectable assembled structure. Thus, it appears that the L75 residue is important for another essential interaction of VP5 with the capsid shell proteins. The congruence of the data from the previous and present studies demonstrates the key roles of two regions in the N terminus of this large protein that are crucial for this bimolecular interaction. Thus, residues I27, L35, and F39 comprise the first subdomain and residues L58, L65, L67 and L71 comprise a second subdomain of VP5. These seven hydrophobic residues are important for the interaction of VP5 with the scaffold protein and consequently the formation of an icosahedral shell structure that encloses the viral genome.     

Identification of a discrete intermediate in the assembly/disassembly of physalis mottle tymovirus through mutational analysis.

Assembly intermediates of icosahedral viruses are usually transient and are difficult to identify. In the present investigation, site-specific and deletion mutants of the coat protein gene of physalis mottle tymovirus (PhMV) were used to delineate the role of specific amino acid residues in the assembly of the virus and to identify intermediates in this process. N-terminal 30, 34, 35 and 39 amino acid deletion and single C-terminal (N188) deletion mutant proteins of PhMV were expressed in Escherichia coli. Site-specific mutants H69A, C75A, W96A, D144N, D144N-T151A, K143E and N188A were also constructed and expressed. The mutant protein lacking 30 amino acid residues from the N terminus self-assembled to T=3 particles in vivo while deletions of 34, 35 and 39 amino acid residues resulted in the mutant proteins that were insoluble. Interestingly, the coat protein (pR PhCP) expressed using pRSET B vector with an additional 41 amino acid residues at the N terminus also assembled into T=3 particles that were more compact and had a smaller diameter. These results demonstrate that the amino-terminal segment is flexible and either the deletion or addition of amino acid residues at the N terminus does not affect T=3 capsid assembly. In contrast, the deletion of even a single residue from the C terminus (PhN188Delta1) resulted in capsids that were unstable. These capsids disassembled to a discrete intermediate with a sedimentation coefficent of 19.4 S. However, the replacement of C-terminal asparagine 188 by alanine led to the formation of stable capsids. The C75A and D144N mutant proteins also assembled into capsids that were as stable as the pR PhCP, suggesting that C75 and D144 are not crucial for the T=3 capsid assembly. pR PhW96A and pR PhD144N-T151A mutant proteins failed to form capsids and were present as heterogeneous aggregates. Interestingly, the pR PhK143E mutant protein behaved in a manner similar to the C-terminal deletion protein in forming unstable capsids. The intermediate with an s value of 19.4 S was the major assembly product of pR PhH69A mutant protein and could correspond to a 30mer. It is possible that the assembly or disassembly is arrested at a similar stage in pR PhN188Delta1, pR PhH69A and pR PhK143E mutant proteins.     

Helix formation and the unfolded state of a 52-residue helical protein

A growing class of proteins in biological processes has been found to be unfolded on isolation under normal solution conditions. We have used NMR spectroscopy to characterize the structural and dynamic properties of the unfolded and partially folded states of a 52-residue alanine-rich protein (Ala-14) at temperatures from -5 degrees C to 40 degrees C. At 40 degrees C, alanine residues in Ala-14 adopt phi and psi angles, consistent with a significant ensemble population of polyproline II conformation. Analysis of relaxation rates in the protein reveals that a series of residues, Gln 35-Ala 36-Ala 37-Lys 38-Asp 39-Asp 40-Ala 41-Ala 42, displays slow motional dynamics at both -5 degrees C and 40 degrees C. Temperature-dependent chemical shift changes indicate that this region is the site of helix initiation. The remaining N-terminal residues become increasingly dynamic as they extend from the nucleation site. The C terminus remains dynamic and changes less with temperature, indicating it is relatively unstructured. Ala-14 provides a high-resolution portrait of the unfolded state and the process of helix nucleation and propagation in the absence of tertiary contacts, information that bears on early events in protein folding.     

Mutational Analysis of the Fusion Peptide of Moloney Murine Leukemia Virus Transmembrane Protein p15E

Fusion peptides are hydrophobic sequences located at the N terminus of the transmembrane (TM) envelope proteins of the orthomyxoviruses and paramyxoviruses and several retroviruses. The Moloney murine leukemia virus TM envelope protein, p15E, contains a hydrophobic stretch of amino acids at its N terminus followed by a region rich in glycine and threonine residues. A series of single amino acid substitutions were introduced into this region, and the resulting proteins were examined for their abilities to be properly processed and transported to the cell surface and to induce syncytia in cells expressing the ecotropic receptor. One substitution in the hydrophobic core and several substitutions in the glycine/threonine-rich region that prevented both cell-cell fusion and the transduction of NIH 3T3 cells when incorporated into retroviral vector particles were identified. In addition, one mutation that enhanced the fusogenicity of the resulting envelope protein was identified. The fusion-defective mutants trans dominantly interfered with the ability of the wild-type envelope protein to cause syncytium formation in a cell-cell fusion assay, although no trans-dominant inhibition of transduction was observed. Certain substitutions in the hydrophobic core that prevented envelope protein processing were also found. These data indicate that the N-terminal region of p15E is important both for viral fusion and for the correct processing and cell surface expression of the viral envelope protein.     

Negatively charged residues in the IgM stop-transfer effector sequence regulate transmembrane polypeptide integration

A non-hydrophobic sequence that contributes to the biogenesis of a transmembrane protein is termed a stop-transfer effector (STE). To examine the mechanism of STE-mediated stop-transfer, a series of fusion proteins were constructed containing variants of a putative STE from murine IgM fused to an otherwise translocated hydrophobic sequence. Unexpectedly, the fraction of molecules adopting transmembrane topology was insensitive to many amino acid substitutions within the STE sequence but varied directly with the number of negative charges. Furthermore, when present at the amino terminus of a reporter, mutants were observed that adopted type I (amino terminus lumenal) and type II (amino terminus cytoplasmic) transmembrane topologies, demonstrating that the STE sequence can be located at either side of the endoplasmic reticulum membrane. Our results suggest that recognition of a broad structural feature formed primarily by negatively charged residues within the STE halts translocation and triggers membrane integration, even when the negative charges end up on the cytoplasmic side of the membrane. Since functional STE sequences photocross-link to two membrane proteins not previously identified at the translocon, these unique proteins are presumably involved in recognizing STE sequences and/or facilitating STE function.     

Structural stability and heat-induced conformational change of two complement inhibitors: C4b-binding protein and factor H

The complement inhibitors C4b-binding protein (C4BP) and factor H (FH) both consist of complement control protein (CCP) domains. Here we examined the secondary structure of both proteins by circular dichroism and Fourier-transform infrared technique at temperatures ranging from 30 degrees C-90 degrees C. We found that predominantly beta-sheet structure of both proteins was stable up to 70 degrees C, and that a reversible conformational change toward alpha-helix was apparent at temperatures ranging from 70 degrees C to 90 degrees C. The ability of both proteins to inhibit complement was not impaired after incubation at 95 degrees C, exposure to extreme pH conditions, and storage at room temperature for several months. Similar remarkable stability was previously observed for vaccinia virus control protein (VCP), which is also composed of CCP domains; it therefore seems to be a general property of CCP-containing proteins. A typical CCP domain has a hydrophobic core, which is wrapped in beta-sheets and stabilized by two disulphide bridges. How the CCP domains tolerate harsh conditions is unclear, but it could be due to a combination of high content of prolines, hydrophobic residues, and the presence of two disulphide bridges within each domain. These findings are of interest because CCP-containing complement inhibitors have been proposed as clinical agents to be used to control unwanted complement activation that contributes to many diseases.     

Matrix protein of Akv murine leukemia virus: genetic mapping of regions essential for particle formation.

Type C retroviruses assemble at the plasma membrane of the infected cell. Attachment of myristic acid to the N terminus of the Gag precursor polyprotein has been shown to be essential for membrane localization and virus morphogenesis. Here, we report that the matrix (MA) protein contains regions that in conjunction with myristylation are important for Gag protein stability and the assembly of murine leukemia viruses. We identified these domains by generating a series of Akv murine leukemia virus mutants carrying small in-frame deletions within the coding region of the MA protein encompassing 129 amino acids. Studies show that mutants with deletions within the segment encoding the first 102 amino acids were all replication defective, whereas the C-terminal residues 103 to 124 seem not to have any critical function in virus maturation. Cells expressing the replication-defective genomes did not release any detectable Gag proteins. In one mutant, deletion of 3 amino acids in the N terminus resulted in an inefficiently myristylated, stable Gag polyprotein. The remaining defect genomes encoded unstable Gag proteins, although they were modified with myristic acid. The results suggest that the matrix domain plays an important role in stabilizing the Gag polyprotein.     

Interaction of PDZRhoGEF with microtubule-associated protein 1 light chains: link between microtubules, actin cytoskeleton, and neuronal polarity

Rat (r) PDZRhoGEF, initially identified as a glutamate transporter EAAT4-associated protein, is a member of a novel RhoGEF subfamily. The N terminus of the protein contains a PDZ and a proline-rich domain, two motifs known to be involved in protein-protein interactions. By using the yeast two-hybrid approach, we screened for proteins that interact with the N terminus of rPDZRhoGEF. The light chain 2 of microtubule-associated protein 1 (LC2) was the only protein identified from the screen that does not contain a type I PDZ-binding motif at its extreme C terminus (-(S/T)Xphi-COOH, where phi is a hydrophobic amino acid). However, the C terminus does conform to a type II-binding motif (-phiXphi). We report here that rPDZRhoGEF interacts with LC2 via the PDZ domain, and the interaction is abolished by mutations in the carboxylate-binding loop. The specificity of the interaction was confirmed using GST fusion protein pull-down assays and coimmunoprecipitations. Expression of rPDZRhoGEF mutants that are unable to interact with proteins via the carboxylate-binding loop induced changes in cell morphology and actin organization. These mutants alter the activation of RhoGTPases, and coexpression of dominant-negative RhoGTPases prevent the morphological changes. Furthermore, in cells expressing wild type rPDZRhoGEF, drug-induced microtubule depolymerization produces changes in cell morphology that are similar to those induced by rPDZRhoGEF mutants. These results indicate that modulation of the guanine nucleotide exchange activity of rPDZRhoGEF through interaction with microtubule-associated protein light chains may coordinate microtubule integrity and the reorganization of actin cytoskeleton. This coordinated action of the actin and microtubular cytoskeletons is essential for the development and maintenance of neuronal polarity.     

Repacking of hydrophobic residues in a stable mutant of apocytochrome b562 selected by phage-display and proteolysis

To test a hydrophobic core-directed protein design approach, we previously have used phage-display and proteolysis to select stably folded proteins from a library of mutants of apocytochrome b562. The consensus sequence of the selected mutants has hydrophilic residues at two of the three positions that are designed to form a hydrophobic core. To understand this unexpected result, we determined the high-resolution structure of one of the selected mutants using multi-dimensional nuclear magnetic resonance (NMR). The structure shows that the two hydrophilic residues in the consensus sequence were on the surface of the structure. Instead, two of their neighboring hydrophobic residues reorganized their side-chain conformations and formed the hydrophobic core. This result suggests that the hydrophobic core-directed protein design by phage-display and proteolysis is a valid method in general but alternative hydrophobic packing needs to be considered in the initial design. The unexpected repacking of the hydrophobic residues also highlights the plastic nature of protein structures.     

Polar residues in the protein core of Escherichia coli thioredoxin are important for fold specificity

Most globular proteins contain a core of hydrophobic residues that are inaccessible to solvent in the folded state. In general, polar residues in the core are thermodynamically unfavorable except when they are able to form intramolecular hydrogen bonds. Compared to hydrophobic interactions, polar interactions are more directional in character and may aid in fold specificity. In a survey of 263 globular protein structures, we found a strong positive correlation between the number of polar residues at core positions and protein size. To probe the importance of buried polar residues, we experimentally tested the effects of hydrophobic mutations at the five polar core residues in Escherichia coli thioredoxin. Proteins with single hydrophobic mutations (D26I, C32A, C35A, T66L, and T77V) all have cooperative unfolding transitions like the wild type (wt), as determined by chemical denaturation. Relative to wt, D26I is more stable while the other point mutants are less stable. The combined 5-fold mutant protein (IAALV) is less stable than wt and has an unfolding transition that is substantially less cooperative than that of wt. NMR spectra as well as amide deuterium exchange indicate that IAALV is likely sampling a number of low-energy structures in the folded state, suggesting that polar residues in the core are important for specifying a well-folded native structure.     

Involvement of a protein tyrosine kinase in production of the polymeric bioemulsifier emulsan from the oil-degrading strain Acinetobacter lwoffii RAG-1

The genes associated with the biosynthesis of the polymeric bioemulsifier emulsan, produced by the oil-degrading Acinetobacter lwoffii RAG-1 are clustered within a 27-kbp region termed the wee cluster. This report demonstrates the involvement of two genes of the wee cluster of RAG-1, wzb and wzc, in emulsan biosynthesis. The two gene products, Wzc and Wzb were overexpressed and purified. Wzc exhibited ATP-dependent autophosphorylating protein tyrosine kinase activity. Wzb was found to be a protein tyrosine phosphatase capable of dephosphorylating the phosphorylated Wzc. Using the synthetic substrate p-nitrophenyl phosphate (PNPP) Wzb exhibited a V(max) of 12 micromol of PNPP min(-1) mg(-1) and a K(m) of 8 mM PNPP at 30 degrees C. The emulsifying activity of mutants lacking either wzb or wzc was 16 and 15% of RAG-1 activity, respectively, suggesting a role for the two enzymes in emulsan production. Phosphorylation of Wzc was found to occur within a cluster of five tyrosine residues at the C terminus. Colonies from a mutant in which these five tyrosine residues were replaced by five phenylalanine residues along with those of a second mutant, which also lacked Wzb, exhibited a highly viscous colony consistency. Emulsan activity of these mutants was 25 and 24% of that of RAG-1, respectively. Neither of these mutants contained cell-associated emulsan. However, they did produce an extracellular high-molecular-mass galactosamine-containing polysaccharide. A model is proposed in which subunit polymerization, translocation and release of emulsan are all associated and coregulated by tyrosine phosphorylation.     

The active N-terminal region of p67phox. Structure at 1.8 A resolution and biochemical characterizations of the A128V mutant implicated in chronic granulomatous disease

Upon activation, the NADPH oxidase from neutrophils produces superoxide anions in response to microbial infection. This enzymatic complex is activated by association of its cytosolic factors p67(phox), p47(phox), and the small G protein Rac with a membrane-associated flavocytochrome b(558). Here we report the crystal structure of the active N-terminal fragment of p67(phox) at 1.8 A resolution, as well as functional studies of p67(phox) mutants. This N-terminal region (residues 1-213) consists mainly of four TPR (tetratricopeptide repeat) motifs in which the C terminus folds back into a hydrophobic groove formed by the TPR domain. The structure is very similar to that of the inactive truncated form of p67(phox) bound to the small G protein Rac previously reported, but differs by the presence of a short C-terminal helix (residues 187-193) that might be part of the activation domain. All p67(phox) mutants responsible for Chronic Granulomatous Disease (CGD), a severe defect of NADPH oxidase function, are localized in the N-terminal region. We investigated two CGD mutations, G78E and A128V. Surprisingly, the A128V CGD mutant is able to fully activate the NADPH oxidase in vitro at 25 degrees C. However, this point mutation represents a temperature-sensitive defect in p67(phox) that explains its phenotype at physiological temperature.     

Functional analysis of the putative fusion domain of the baculovirus envelope fusion protein F

Group II nucleopolyhedroviruses (NPVs), e.g., Spodoptera exigua MNPV, lack a GP64-like protein that is present in group I NPVs but have an unrelated envelope fusion protein named F. In contrast to GP64, the F protein has to be activated by a posttranslational cleavage mechanism to become fusogenic. In several vertebrate viral fusion proteins, the cleavage activation generates a new N terminus which forms the so-called fusion peptide. This fusion peptide inserts in the cellular membrane, thereby facilitating apposition of the viral and cellular membrane upon sequential conformational changes of the fusion protein. A similar peptide has been identified in NPV F proteins at the N terminus of the large membrane-anchored subunit F(1). The role of individual amino acids in this putative fusion peptide on viral infectivity and propagation was studied by mutagenesis. Mutant F proteins with single amino acid changes as well as an F protein with a deleted putative fusion peptide were introduced in gp64-null Autographa californica MNPV budded viruses (BVs). None of the mutations analyzed had an major effect on the processing and incorporation of F proteins in the envelope of BVs. Only two mutants, one with a substitution for a hydrophobic residue (F152R) and one with a deleted putative fusion peptide, were completely unable to rescue the gp64-null mutant. Several nonconservative substitutions for other hydrophobic residues and the conserved lysine residue had only an effect on viral infectivity. In contrast to what was expected from vertebrate virus fusion peptides, alanine substitutions for glycines did not show any effect.