SorCS3 does not require propeptide cleavage to bind nerve growth factor

The functional properties of the Vps10p-domain receptor SorCS3 are undescribed. Here, we examine its processing and sorting in cellular transfectants, and analyze the binding of potential ligands to the purified receptor. We show that SorCS3 is synthesized as a proprotein and converted to its mature form by N-terminal propeptide cleavage in distal Golgi compartments. The propeptide is not a requirement for normal processing of the receptor and does not prevent ligands from binding to the SorCS3 precursor form. Expression of wt and chimeric receptors further suggests that SorCS3 predominates on the plasma membrane, exhibits slow internalization and does not engage in intracellular trafficking. SorCS3 emerges as a new neurotrophin binding Vps10p-domain receptor functionally distinct from its relatives Sortilin and SorLA.     

Signal sequence recognition and targeting of ribosomes to the endoplasmic reticulum by the signal recognition particle do not require GTP.

The identification of GTP-binding sites in the 54-kDa subunit of the signal recognition particle (SRP) and in both the alpha and beta subunits of the SRP receptor has complicated the task of defining the step in the protein translocation reaction that is controlled by the GTP-binding site in the SRP. Ribonucleotide binding assays show that the purified SRP can bind GDP or GTP. However, crosslinking experiments show that SRP54 can recognize the signal sequence of a nascent polypeptide in the absence of GTP. Targeting of SRP-ribosome-nascent polypeptide complexes, formed in the absence of GTP, to microsomal membranes likewise proceeds normally. To separate the GTPase cycles of SRP54 and the alpha subunit of the SRP receptor (SR alpha), we employed an SR alpha mutant that displays a markedly reduced affinity for GTP. We observed that the dissociation of SRP54 from the signal sequence and the insertion of the nascent polypeptide into the translocation site could only occur when GTP binding to SR alpha was permitted. These data suggest that the GTP binding and hydrolysis cycles of both SRP54 and SR alpha are initiated upon formation of the SRP-SRP receptor complex.     

T cell recognition of fibrinogen. A determinant on the A alpha-chain does not require processing

Murine T cell hybridomas were used to examine the requirements for processing and presentation of human fibrinogen. In contrast to most protein Ag, fibrinogen (Mr 340,000) did not need to be processed by an APC, inasmuch as intact fibrinogen was presented by both pre-fixed and chloroquine-treated macrophages. Through the use of a variety of protease inhibitors, no involvement of proteases either on the macrophage surface or in the culture medium in the presentation of fibrinogen was observed. The epitope recognized by two T cell hybridomas was localized to the peptide, A alpha (551-578), which was located on the carboxy portion of the A alpha-chain. This portion of the A alpha-chain has no defined secondary structure and must possess the conformational flexibility which allows it to directly associate with an I-Ek molecule. Thus conformational flexibility may be a major factor in determining the processing requirements of a protein Ag.     

SecB functions as a cytosolic signal recognition factor for protein export in E. coli

A purified 64 kd protein, consisting of four identical subunits of the 16 kd SecB, binds to the signal sequence of preproteins prior to their translocation across inverted vesicles (INV) derived from the E. coli plasma membrane. The purified SecB tetramer competes with canine signal recognition particle (SRP) in signal sequence binding and thus behaves as a prokaryotic equivalent of SRP. As shown by cell fractionation and immunoblot analysis with anti-SecB antibodies, SecB is a cytosolic protein. An E. coli supernatant depleted of SecB after passage through an anti-SecB Sepharose column retains full translation activity but is unable to support translocation into added INV. Translocation into INV is fully restored by readdition of purified SecB.     

The signal recognition particle-targeting pathway does not necessarily deliver proteins to the sec-translocase in Escherichia coli.

ProW is an Escherichia coli inner membrane protein that consists of a 100-residue-long periplasmic N-terminal tail (N-tail) followed by seven closely spaced transmembrane segments. N-tail translocation presumably proceeds in a C-to-N-terminal direction and represents a poorly understood aspect of membrane protein biogenesis. Here, using an in vivo depletion approach, we show that N-tail translocation in a ProW derivative comprising the N-tail and the first transmembrane segment fused to the globular P2 domain of leader peptidase depends both on the bacterial signal recognition particle (SRP) and the Sec-translocase. Surprisingly, however, a deletion construct with only one transmembrane segment downstream of the N-tail can assemble properly even under severe depletion of SecE, a central component of the Sec-translocase, but not under SRP-depletion conditions. To our knowledge, this is the first demonstration that the SRP-targeting pathway does not necessarily deliver SRP-dependent inner membrane proteins to the Sec-translocase. The data further suggest that N-tail translocation can proceed in the absence of a functional Sec-translocase.     

Entry of poliovirus into cells does not require a low-pH step.

The requirement of a low-pH step during poliovirus entry was investigated by using the macrolide antibiotic bafilomycin A1, which is a powerful and selective inhibitor of the vacuolar proton-ATPases. Thus, viruses such as Semliki Forest virus and vesicular stomatitis virus that enter cells through endosomes and need their acidification, are potently inhibited by bafilomycin A1, whereas poliovirus infection is not affected by the antibiotic. The presence of lysosomotropic agents such as chloroquine, amantadine, dansylcadaverine, and monensin during poliovirus entry did not inhibit infection, further supporting the idea that poliovirus does not depend on a low-pH step to enter the cytoplasm. The effect of bafilomycin A1 on other members of the Picornaviridae family was also assayed. Encephalomyocarditis virus entry into HeLa cells was not affected by the macrolide antibiotic, whereas rhinovirus was sensitive. Coentry of toxins, such as alpha-sarcin, with viral particles was potently inhibited by bafilomycin A1, indicating that an active vacuolar proton-ATPase is necessary for the early membrane permeabilization (coentry of alpha-sarcin) induced by poliovirus to take place.     

Human complement factor I does not require cofactors for cleavage of synthetic substrates

Complement factor I (fI) plays a major role in the regulation of the complement system. It circulates in an active form and has very restricted specificity, cleaving only C3b or C4b in the presence of a cofactor such as factor H (fH), complement receptor type 1, membrane cofactor protein, or C4-binding protein. Using peptide-7-amino-4-methylcoumarin derivatives, we investigated the substrate specificity of fI. There is no previous report of synthetic substrate cleavage by fI, but five substrates were found in this study. A survey of 15 substrates and a range of inhibitors showed that fI has specificity similar to that of thrombin, but with much lower catalytic activity than that of thrombin. fI amidolytic activity has a pH optimum of 8.25, typical of serine proteases and is insensitive to ionic strength. This is in contrast to its proteolytic activity within the fI-C3b-fH reaction, in which the pH optimum for C3b cleavage is <5.5 and the reaction rate is highly dependent on ionic strength. The rate of cleavage of tripeptide 7-amino-4-methylcoumarins by fI is unaffected by the presence of fH or C3(NH(3)). The amidolytic activity is inhibited by the synthetic thrombin inhibitor Z-D-Phe-Pro-methoxypropylboroglycinepinanediol ester, consistent with previous reports, and by benzenesulfonyl fluorides such as Pefabloc SC. Suramin inhibits fI directly at concentration of 1 mM. Within a range of metal ions tested, only Cr(2+) and Fe(3+) were found to inhibit both the proteolytic and amidolytic activity of fI.     

Inositol phosphate formation in fMet-Leu-Phe-stimulated human neutrophils does not require an increase in the cytosolic free Ca2+ concentration.

The accumulation of inositol phosphates in myo-[3H]inositol-labelled human neutrophils stimulated with the chemotactic peptide fMet-Leu-Phe was measured. The challenge with the chemotactic peptide caused the generation of inositol monophosphate (InsP), inositol bisphosphate (InsP2) and inositol trisphosphate (InsP3). The formation of the three inositol phosphates followed a differential time course: InsP3 accumulated very rapidly and transiently, whereas InsP increased steadily for more than 2 min. Inositol phosphate formation was only partially decreased by procedures which prevented the fMet-Leu-Phe-dependent increase of cytosolic free Ca2+ concentration.     

Adenovirus homologous recombination does not require expression of the immediate-early E1a gene.

To investigate whether early genes other than those involved directly in DNA replication are required for efficient adenovirus recombination, pairs of viruses with deletions in E1a, E1b 496R, E1b 196R, or E4 and containing differing restriction site markers were used to infect both permissive and non- or semipermissive cells. Recombination was assayed among intracellular and extracellular genomes by restriction digestion and blot hybridization. Recombination was delayed in infections of nonpermissive cells with E1a- viruses until a time consistent with the late onset of DNA replication characteristic of the cell type. This shows that E1a expression is not absolutely required for adenovirus recombination. Similar tests with deletion mutations in E1b and E4 also show that these genes are not required for efficient recombination. Taken together with earlier results showing that recombination depends on DNA replication, it is likely that adenovirus recombination is a consequence of cellular repair functions acting on the substrates produced by replication.     

Malignant transformation of murine fibroblasts by a human c-Ha-ras-1 oncogene does not require a functional epidermal growth factor receptor.

Although mutations in ras genes are thought to be important for the development of about 20% of human tumors, almost nothing is known about the way in which these mutations lead to cellular transformation. The known biochemical properties of the 21-kilodalton ras proteins suggest that they may behave as G proteins, regulating the proliferation of cells in response to growth factor stimulation of a receptor. Although the putative receptor(s) has not been identified, several lines of evidence, in particular the fact that rodent cell lines containing ras oncogenes produce transforming growth factor alpha, have suggested that the epidermal growth factor (EGF) receptor is involved in ras transformation. Here we show that murine fibroblasts with no EGF receptors can be transformed to a completely malignant phenotype with a mutated ras gene. It appears, therefore, that the EGF receptor is not required for ras-mediated transformation of these cells.     

Targeting proteins to membranes: structure of the signal recognition particle

In all three kingdoms of life, co-translational targeting of secretory and membrane proteins to the prokaryotic plasma membrane or eukaryotic endoplasmic reticulum is mediated by a ribonucleoprotein complex, the signal recognition particle (SRP), and its membrane-associated receptor (SR). SRP binds to signal sequences of nascent proteins as they emerge from the exit tunnel of the ribosome. The resulting targeting complex, composed of the SRP and the ribosome-nascent chain complex (RNC), then docks with the SR in a GTP-dependent manner. Passing through a complex series of conformational states, SRP and SR deliver the RNC to the translocon, which in turn mediates protein translocation across or integration into the membrane. The core structural and mechanistic principles of SRP-dependent protein targeting are universally conserved. Recent structural investigations combining X-ray crystallography and cryo-electron microscopy have provided new insights into three essentials steps of the SRP-dependent protein targeting cycle: the assembly and interaction of the SRP ribonucleoprotein core, the GTP-dependent SRP-SR association, and the interaction between SRP and the ribosome.     

Induction of lamellipodia by Kalirin does not require its guanine nucleotide exchange factor activity

Guanine nucleotide exchange factor (GEF) domains of the Dbl family occur in a variety of proteins that include multiple protein-protein and protein-lipid interaction domains. We used an epithelial-derived cell line to investigate the mechanisms by which the two GEF domains of Kalirin, a neuronal Rho GEF, influence morphology. As expected, Kal-GEF1, an efficient GEF for Rac1 and RhoG, induced the formation of lamellipodia resembling those induced by active Rac1. Although Kal-GEF1 activated Rac and Pak, its ability to induce formation of lamellipodia was not blocked by dominant negative Rho GTPases or by catalytically inactive Pak. Consistent with this, a catalytically inactive mutant of Kal-GEF1 induced formation of lamellipodia and activated Pak. Active Pak was required for the GEF-activity independent effect of Kal-GEF1 and the lamellipodia produced were filled with ribs of filamentous actin. Kal-GEF1 and a GEF-dead mutant co-immunoprecipitated with Pak. The interaction of Kal-GEF1 with Pak is indirect and requires the regulatory protein binding domain of Pak. Filamin A, which is known to interact with and activate Pak, binds to both catalytically active and inactive Kal-GEF1, providing a link by which catalytically inactive Kal-GEF1 can activate Pak and induce lamellipodia. Together, our results indicate that Kal-GEF1 induces lamellipodia through activation of Pak, where GEF activity is not required. GEF-activity-independent effects on downstream targets may be a general property of RhoGEFs.     

Targeted deletion of the cytosolic domain of tissue factor in mice does not affect development

The role of the cytosolic domain of tissue factor (TF) in signal transduction and gene regulation was studied in mice with a targeted deletion of the 18 carboxy-terminal intracellular amino acids. This deletion was introduced in exon 6 along with a floxed neo(R) selection cassette in intron 5 using homologous recombination in embryonic stem cells. Removal of the floxed neo(R) cassette by in vivo Cre-mediated loxP recombination yielded TF(+/deltaCT) and TF(deltaCT/deltaCT) mice. In contrast to TF(-/-) mice, TF(+/deltaCT) and TF(deltaCT/deltaCT) mice displayed normal embryonic development, survival, fertility, and blood coagulation. Factor VIIa or factor Xa stimulation produced similar p44/42 MAPK activation in TF(+/+) and TF(deltaCT/deltaCT) fibroblasts. These data, based on expression of a TF(deltaCT) molecule from the endogenous TF locus, provide conclusive proof that the cytosolic domain of TF is not essential for signal transduction in embryogenesis and in physiological postnatal processes.     

Topogenesis of peroxisomal proteins does not require a functional cytoplasm-to-vacuole transport

Folded and even oligomeric proteins can be imported from the cytosol into vacuoles and into peroxisomes. Pro-aminopeptidase I (prAPI) oligomerizes into a dodecamer and is imported into the vacuole via the cytoplasm-to-vacuole transport (cvt) pathway. How peroxisomes accommodate folded proteins is completely unknown. Peroxisome biogenesis and cvt do not only share the import of folded protein complexes but also show mechanistic parallels such as the employment of ubiquitin conjugation systems. In search for a genetic overlap, selected cvt and autophagocytosis (atg) mutants were tested for defects in peroxisome biogenesis. Most of the mutants did not exhibit a mislocalization of peroxisomal matrix proteins to the cytosol which would be typical of a defect in the peroxisome biogenesis. However, two mutants, deltaatg14 and deltacvt4/vam6, displayed a general growth defect and deltacvt8/vps41 showed cytosolic mislocalization not only of peroxisomal but also of mitochondrial proteins, indicating a more general defect in organelle biogenesis. Our data do not provide evidence for a genetic overlap of the import pathway for peroxisomal proteins and the cvt pathway.     

OxLDL-mediated survival of macrophages does not require LDL internalization or signalling by major pattern recognition receptors

Macrophages play a key role in the pathogenesis of atherosclerosis, in part by destabilizing plaques. We and others have shown that low concentrations of oxidized LDL (oxLDL) inhibit macrophage apoptosis. As oxLDL is present in lesions, this may be a mechanism by which macrophage populations in the intima are expanded. We have previously shown that oxLDL activates prosurvival signalling pathways such as the phosphoinositide 3-kinase (PI3K) pathway in bone marrow derived macrophages (BMDMs). However, little is known about more upstream signalling events especially at the receptor level. The endocytic pattern recognition receptors (PRRs), scavenger receptor A (SR-A) and CD36, are the main receptors on macrophages for uptake of oxLDL and are therefore important in foam cell formation. The signalling PRRs such as toll-like receptor (TLR) 2 and 4 also bind some types of oxLDL. This study was done to determine if any of the known PRRs are required for the anti-apoptotic effects of oxLDL in BMDMs. To do this, we tested the effect of oxLDL on viability of BMDMs lacking both SR-A and CD36 or lacking TLR2, TLR4, CD14, FcγRIIb, or RAGE. Our results indicate that none of these receptors are essential for activating the oxLDL prosurvival pathway. Furthermore, we show that the anti-apoptotic effect is not dependent on the uptake of oxLDL.     

Transmembrane kit ligand cleavage does not require a signal in the cytoplasmic domain and occurs at a site dependent on spacing from the membrane.

The kit ligand (KL) is one of several growth factors that are active as transmembrane molecules and can also be proteolytically cleaved to yield soluble forms. We have investigated the signals and structural determinants that control the cleavage of KL. Presentation at the membrane appears to be critical, because no cleavage occurs in variants that lack a transmembrane domain. Signals in the cytoplasmic domain do not seem to be required, because cleavage was not blocked by removal of the C-terminal valine residue, deletion of the whole cytoplasmic tail, or the replacement of the cytoplasmic tail that occurs in the Sl17H mutation. KL thus appears to differ from transforming growth factor-alpha, which apparently requires a C-terminal valine as a signal for cleavage. Although proteolysis must be tightly restricted to the correct cell surface proteins and sites within each protein, cleavage of KL does not seem to be determined entirely by a requirement for a specific substrate sequence. However, the effects of deletion or insertion variants of KL suggest that cleavage may be limited to sites within a specific range of distances from the cell membrane.     

Targeting of a cytosolic protein to the nuclear periphery

The yeast nuclear envelope protein NSP1 is located at the nuclear pores and mediates its essential function via the carboxy-terminal domain. The passenger protein, cytosolic dihydrofolate reductase from mouse, was fused to the 220 residue long NSP1 carboxy-terminal domain. When expressed in yeast, this chimeric protein was tightly associated with nuclear structures and was localized at the nuclear periphery very similar to authentic NSP1. Furthermore, the DHFR-C-NSP1 fusion protein was able to complement a yeast mutant lacking a functional NSP1 gene showing that DHFR-C-NSP1 fulfils the same basic function as compared to the endogenous NSP1 protein. These data also show that the NSP1 protein is composed of separate functional moieties: a carboxy-terminal domain that is sufficient to mediate the association with the nuclear periphery and an amino-terminal and middle repetitive domain with an as yet unknown function. It is suggested that heptad repeats found in the NSP1 carboxy-terminal domain, which are similar to those found in intermediate filament proteins, are crucial for mediating the association with the nuclear pores.     

Replicative aging of the yeast does not require DNA replication

Glucose addition to a stationary culture of wild-type Saccharomyces cerevisiae BY4742 cells with zero activity of MDR pumps resuspended in a fresh medium causes pump resynthesis (measured as pump-effected diS-C3(3) efflux). In a stationary culture in its original growth medium, this glucose-induced pump resynthesis fails to occur due to depletion of essential nutrients or to extracellular metabolites produced by cells during growth. Direct pump inactivation by metabolites is excluded since exponential cells with high MDR pump activity cultured in a medium with high concentration of extracellular metabolites retain this activity for at least 2 h. The metabolites also do not affect pump synthesis on the level of gene expression as addition of concentrated growth medium or an amino acid mixture to stationary cells in spent growth medium restores glucose-induced pump synthesis. The block of MDR pump synthesis is therefore due to the lack of essential nutrients in spent medium.