The C-terminal domain of TRPV4 is essential for plasma membrane localization

Many members of the TRP superfamily oligomerize in the ER before trafficking to the plasma membrane. For membrane localization of the non-selective cation channel TRPV4 specific domains in the N-terminus are required, but the role of the C-terminus in the oligomerization and trafficking process has been not determined until now. Therefore, the localization of recombinant TRPV4 in two cell models was analyzed: HaCaT keratinocytes that express TRPV4 endogenously were compared to CHO cells that are devoid of endogenous TRPV4. When deletions were introduced in the C-terminal domain three states of TRPV4 localization were defined: a truncated TRPV4 protein of 855 amino acids was exported to the plasma membrane like the full-length channel (871 aa) and was also functional. Mutants with a length of 828 to 844 amino acids remained in the ER of CHO cells, but in HaCaT cells plasma membrane localization was partially rescued by oligomerization with endogenous TRPV4. This was confirmed by coexpression of recombinant full-length TRPV4 together with these deletion mutants, which resulted in an almost complete plasma membrane localization of both proteins and significant FRET in the plasma membrane and the ER. All deletions upstream of amino acid 828 resulted in total ER retention that could not rescued by coexpression with the full-length protein. However, these deletion mutants did not impair export of full-length TRPV4, implying that no oligomerization took place. These data indicate that the C-terminus of TRPV4 is required for oligomerization, which takes place in the ER and precedes plasma membrane trafficking.     

The C-terminal domain of TRPV4 is essential for plasma membrane localization

Many members of the TRP superfamily oligomerize in the ER before trafficking to the plasma membrane. For membrane localization of the non-selective cation channel TRPV4 specific domains in the N-terminus are required, but the role of the C-terminus in the oligomerization and trafficking process has been not determined until now. Therefore, the localization of recombinant TRPV4 in two cell models was analyzed: HaCaT keratinocytes that express TRPV4 endogenously were compared to CHO cells that are devoid of endogenous TRPV4. When deletions were introduced in the C-terminal domain three states of TRPV4 localization were defined: a truncated TRPV4 protein of 855 amino acids was exported to the plasma membrane like the full-length channel (871 aa) and was also functional. Mutants with a length of 828 to 844 amino acids remained in the ER of CHO cells, but in HaCaT cells plasma membrane localization was partially rescued by oligomerization with endogenous TRPV4. This was confirmed by coexpression of recombinant full-length TRPV4 together with these deletion mutants, which resulted in an almost complete plasma membrane localization of both proteins and significant FRET in the plasma membrane and the ER. All deletions upstream of amino acid 828 resulted in total ER retention that could not rescued by coexpression with the full-length protein. However, these deletion mutants did not impair export of full-length TRPV4, implying that no oligomerization took place. These data indicate that the C-terminus of TRPV4 is required for oligomerization, which takes place in the ER and precedes plasma membrane trafficking.     

A short C-terminal domain of Yku70p is essential for telomere maintenance

The Yku heterodimer from Saccharomyces cerevisiae, comprising Yku70p and Yku80p, is involved in the maintenance of a normal telomeric DNA end structure and is an essential component of nonhomologous end joining (NHEJ). To investigate the role of the Yku70p subunit in these two different pathways, we generated C-terminal deletions of the Yku70 protein and examined their ability to complement the phenotypes of a yku70(-) strain. Deleting only the 30 C-terminal amino acids of Yku70p abolishes Yku DNA binding activity and causes a yku(-) phenotype; telomeres are shortened, and NHEJ is impaired. Using conditions in which at least as much mutant protein as full-length protein is normally detectable in cell extracts, deleting only 25 C-terminal amino acids of Yku70p results in no measurable effect on DNA binding of the Yku protein, and the cells are fully proficient for NHEJ. Nevertheless, these cells display considerably shortened telomeres, and significant amounts of single-stranded overhangs of the telomeric guanosine-rich strands are observed. Co-overexpression of this protein with Yku80p could rescue some but not all of the telomere-related phenotypes. Therefore, the C-terminal domain in Yku70p defines at least one domain that is especially involved in telomere maintenance but not in NHEJ.     

A C-terminal silencing domain in GW182 is essential for miRNA function

Proteins of the GW182 family are essential for miRNA-mediated gene silencing in animal cells; they interact with Argonaute proteins (AGOs) and are required for both the translational repression and mRNA degradation mediated by miRNAs. To gain insight into the role of the GW182–AGO1 interaction in silencing, we generated protein mutants that do not interact and tested them in complementation assays. We show that silencing of miRNA targets requires the N-terminal domain of GW182, which interacts with AGO1 through multiple glycine–tryptophan (GW)-repeats. Indeed, a GW182 mutant that does not interact with AGO1 cannot rescue silencing in cells depleted of endogenous GW182. Conversely, silencing is impaired by mutations in AGO1 that strongly reduce the interaction with GW182 but not with miRNAs. We further show that a GW182 mutant that does not localize to P-bodies but interacts with AGO1 rescues silencing in GW182-depleted cells, even though in these cells, AGO1 also fails to localize to P-bodies. Finally, we show that in addition to the N-terminal AGO1-binding domain, the middle and C-terminal regions of GW182 (referred to as the bipartite silencing domain) are essential for silencing. Together our results indicate that miRNA silencing in animal cells is mediated by AGO1 in complex with GW182, and that P-body localization is not required for silencing.     

Crystal structure of yeast mitochondrial peripheral membrane protein Tim44p C-terminal domain

The protein transports from the cell cytosol to the mitochondria matrix are carried out by the translocase of the outer membrane (TOM) complex and the translocase of the inner membrane (TIM) complexes. Tim44p is an essential mitochondrial peripheral membrane protein and a major component of TIM23 translocon. Tim44p can tightly associate with the inner mitochondrial membrane. To investigate the mechanism by which Tim44p functions in the TIM23 translocon to deliver the mitochondrial protein precursors, we have determined the crystal structure of the yeast Tim44p C-terminal domain to 3.2A resolution using the MAD method. The Tim44p C-terminal domain forms a monomer in the crystal structure and contains six alpha-helices and four antiparallel beta-strands. A large hydrophobic pocket was identified on the Tim44p structure surface. The N-terminal helix A1 is positively charged and the helix A1 protrudes out from the Tim44p main body.     

The C-terminal flexible domain of the heme chaperone CcmE is important but not essential for its function

CcmE is a heme chaperone active in the cytochrome c maturation pathway of Escherichia coli. It first binds heme covalently to strictly conserved histidine H130 and subsequently delivers it to apo-cytochrome c. The recently solved structure of soluble CcmE revealed a compact core consisting of a beta-barrel and a flexible C-terminal domain with a short alpha-helical turn. In order to elucidate the function of this poorly conserved domain, CcmE was truncated stepwise from the C terminus. Removal of all 29 amino acids up to crucial histidine 130 did not abolish heme binding completely. For detectable transfer of heme to type c cytochromes, only one additional residue, D131, was required, and for efficient cytochrome c maturation, the seven-residue sequence (131)DENYTPP(137) was required. When soluble forms of CcmE were expressed in the periplasm, the C-terminal domain had to be slightly longer to allow detection of holo-CcmE. Soluble full-length CcmE had low activity in cytochrome c maturation, indicating the importance of the N-terminal membrane anchor for the in vivo function of CcmE.     

The Survivin-Crm1 interaction is essential for chromosomal passenger complex localization and function

The chromosomal passenger complex (CPC) of Aurora-B, Borealin, INCENP (inner centromere protein) and Survivin coordinates essential chromosomal and cytoskeletal events during mitosis. Here, we show that the nuclear export receptor Crm1 is crucially involved in tethering the CPC to the centromere by interacting with a leucine-rich nuclear export signal (NES), evolutionarily conserved in all mammalian Survivin proteins. We show that inhibition of the Survivin-Crm1 interaction by treatment with leptomycin B or by RNA-interference-mediated Crm1 depletion prevents centromeric targeting of Survivin. The genetic inactivation of the Survivin-Crm1 interaction by mutation of the NES affects the correct localization and function of Survivin and the CPC during mitosis. By contrast, CPC assembly does not seem to require the Survivin-Crm1 interaction. Our report shows the functional significance of the Survivin-Crm1 interface and provides a novel link between the mitotic effector Crm1 and the CPC.     

Interaction of the yeast gamma-tubulin complex-binding protein Spc72p with Kar1p is essential for microtubule function during karyogamy.

The spindle pole body component Kar1p has a function in nuclear fusion during conjugation, a process known as karyogamy. The molecular role of Kar1p during this process is poorly understood. Here we show that the yeast gamma-tubulin complex-binding protein Spc72p interacts directly with the N-terminal domain of Kar1p, thereby targeting the gamma-tubulin complex to the half bridge, a substructure of the spindle pole body, where it organizes microtubules. This binding of Spc72p to Kar1p has only a minor role during vegetative growth, whereas it becomes essential for karyogamy in mating cells, explaining the important role of Kar1p in this process. We also show that the localization of Spc72p within the spindle pole body changes throughout the cell cycle and even more strongly in response to mating pheromone. Taken together, these observations suggest that the relocalization of Spc72p within the spindle pole body is the 'landmark' event in the pheromone-induced reorganization of the cytoplasmic microtubules.     

The death domain of NF-kappa B1 p105 is essential for signal-induced p105 proteolysis

Stimulation of cells with tumor necrosis factor alpha (TNFalpha) triggers NF-kappaB1 p105 proteolysis, releasing associated Rel subunits to translocate into the nucleus and modulate target gene expression. Phosphorylation of serine 927 within the p105 PEST region by the IkappaB kinase (IKK) complex is required to promote p105 proteolysis in response to TNFalpha stimulation. In this study, the role of the p105 death domain (DD) in signal-induced p105 proteolysis is investigated. Endogenous p105 is shown to interact with the IKK complex in HeLa cells, and transient transfection experiments in 293 cells indicate that each of the catalytic components of the IKK complex, IKK1 and IKK2, can bind to p105. Interaction of p105 with both IKK1 and IKK2 is substantially reduced by deletion of the p105 DD or introduction of a specific point mutation (L841A) into the p105 DD homologous to the lpr mutation in Fas. Phosphorylation of immunoprecipitated p105 on serine 927 by purified recombinant IKK1 or IKK2 protein in vitro is dramatically reduced in both DD mutants relative to wild type. Furthermore, both of the DD mutations significantly impair the ability of low concentrations of IKK2 to induce p105 serine 927 phosphorylation and proteolysis in transiently transfected 3T3 cells. However, high levels of transiently expressed IKK2 bypass the requirement for the p105 DD to induce p105 serine 927 phosphorylation. Finally, p105 serine 927 phosphorylation by the endogenous IKK complex after TNFalpha stimulation and subsequent p105 proteolysis is blocked in both p105 DD mutants when stably expressed in HeLa cells. Thus, the p105 DD acts as a docking site for IKK, increasing its local concentration in the vicinity of the p105 PEST region and facilitating efficient serine 927 phosphorylation.     

Nuclear localization of yeast Nfs1p is required for cell survival

Saccharomyces cerevisiae Nfs1p is mainly found in the mitochondrial matrix and has been shown to participate in iron-sulfur cluster assembly. We show here that Nfs1p contains a potential nuclear localization signal, RRRPR, in its mature part. When this sequence was mutated to RRGSR, the mutant protein could not restore cell growth under chromosomal NFS1-depleted conditions. However, this mutation did not affect the function of Nfs1p in biogenesis of mitochondrial iron-sulfur proteins. The growth defect of the mutant was complemented by simultaneous expression of the mature Nfs1p, which contains the intact nuclear localization signal but lacks its mitochondrial-targeting presequence. These results suggest that a fraction of Nfs1p is localized in the nucleus and is essential for cell viability.     

The cell death protease Kex1p is essential for hypochlorite-induced apoptosis in yeast

Following microbial pathogen invasion, the human immune system of activated phagocytes generates and releases the potent oxidant hypochlorous acid (HOCl), which contributes to the killing of menacing microorganisms. Though tightly controlled, HOCl generation by the myeloperoxidase-hydrogen peroxide-chloride system of neutrophils/monocytes may occur in excess and lead to tissue damage. It is thus of marked importance to delineate the molecular pathways underlying HOCl cytotoxicity in both microbial and human cells. Here, we show that HOCl induces the generation of reactive oxygen species (ROS), apoptotic cell death and the formation of specific HOCl-modified epitopes in the budding yeast Saccharomyces cerevisiae. Interestingly, HOCl cytotoxicity can be prevented by treatment with ROS scavengers, suggesting oxidative stress to mediate the lethal effect. The executing pathway involves the pro-apoptotic protease Kex1p, since its absence diminishes HOCl-induced production of ROS, apoptosis and protein modification. By characterizing HOCl-induced cell death in yeast and identifying a corresponding central executor, these results pave the way for the use of Saccharomyces cerevisiae in HOCl research, not least given that it combines both being a microorganism as well as a model for programmed cell death in higher eukaryotes.     

The BRCA1 C-terminal domain: structure and function

The BRCA1 C-terminal region contains a duplicated globular domain termed BRCT that is found within many DNA damage repair and cell cycle checkpoint proteins. The unique diversity of this domain superfamily allows BRCT modules to interact forming homo/hetero BRCT multimers, BRCT-non-BRCT interactions, and interactions with DNA strand breaks. The sequence and functional diversity of the BRCT superfamily suggests that BRCT domains are evolutionarily convenient interaction modules.     

Phosphatidyl ethanolamine is essential for targeting the arginine transporter Can1p to the plasma membrane of yeast

In continuation of our previous study, we show that phosphatidyl ethanolamine (PE) depletion affects, in addition to amino acid transporters, activities of at least two other proton motive force (pmf)-driven transporters (Ura4p and Mal6p). For Can1p, we demonstrate that the lack of PE results in a failure of the permease targeting to plasma membrane. Despite the pleiotropic effect of PE depletion, a specific role of PE in secretion of a defined group of permeases can be distinguished. Pmf-driven transporters are more sensitive to the lack of PE than other plasma membrane proteins.     

The amino-terminal domain of yeast U1-70K is necessary and sufficient for function.

The Saccharomyces cerevisiae SNP1 gene encodes a protein that shares 30% amino acid identity with the mammalian U1 small nuclear ribonucleoprotein particle protein 70K (U1-70K). We have demonstrated that yeast strains in which the SNP1 gene was disrupted are viable but exhibit greatly increased doubling times and severe temperature sensitivity. Furthermore, snp1-null strains are defective in pre-mRNA splicing. We have tested deletion alleles of SNP1 for their ability to complement these phenotypes. We found that the highly conserved RNA recognition motif consensus domain of Snp1 is not required for complementation of the snp1-null growth or splicing defects nor for the in vivo association with the U1 small nuclear ribonucleoprotein particle. However, the amino-terminal domain of Snp1, less strongly conserved, is necessary and sufficient for complementation.     

The PH domain of the yeast GEF Rom2p serves an essential function in vivo

In a screen designed to identify new upstream components of the Pkc1p-MAP kinase signal transduction pathway that responds to cell wall damage in yeast, we identified a new mutant allele of the ROM2 gene, which encodes a GDP/GTP exchange factor that acts on Rho1p. This allele, originally termed ubk1 (upstream of Bck1p) encodes a truncated protein that lacks the putative PH domain. Complementation experiments showed that genes coding for several known components of the pathway are able to suppress the ubk1 mutation to various degrees when introduced on low- or high-copy-number vectors. Analysis of several rom2 mutants showed that mutants in which the PH domain is deleted result in a phenotype indistinguishable from that of a strain deleted for the entire gene, indicating that this domain fulfills an essential function in vivo. Furthermore, we found that the growth phenotype of rom2 mutants is highly dependent on the strain background. Surprisingly, analysis of the phosphorylation status of Mpk1p in these mutants showed an elevated level of doubly phosphorylated Mpk1 protein, indicating that the growth defect of rom2 mutants is not due to an inability to activate the MAP kinase module, but rather to lack of a function of the Rom2 protein that has yet to be identified precisely.     

The yeast gene ERG6 is required for normal membrane function but is not essential for biosynthesis of the cell-cycle-sparking sterol.

In Saccharomyces cerevisiae, methylation of the principal membrane sterol at C-24 produces the C-28 methyl group specific to ergosterol and represents one of the few structural differences between ergosterol and cholesterol. C-28 in S. cerevisiae has been suggested to be essential for the sparking function (W. J. Pinto and W. R. Nes, J. Biol. Chem. 258:4472-4476, 1983), a cell cycle event that may be required to enter G1 (C. Dahl, H.-P. Biemann, and J. Dahl, Proc. Natl. Acad. Sci. USA 84:4012-4016, 1987). The sterol biosynthetic pathway in S. cerevisiae was genetically altered to assess the functional role of the C-28 methyl group of ergosterol. ERG6, the putative structural gene for S-adenosylmethionine: delta 24-methyltransferase, which catalyzes C-24 methylation, was cloned, and haploid strains containing erg6 null alleles (erg6 delta 1 and erg6 delta ::LEU2) were generated. Although erg6 delta cells are unable to methylate ergosterol precursors at C-24, they exhibit normal vegatative growth, suggesting that C-28 sterols are not essential in S. cerevisiae. However, erg6 delta cells exhibit pleiotropic phenotypes that include defective conjugation, hypersensitivity to cycloheximide, resistance to nystatin, a severely diminished capacity for genetic transformation, and defective tryptophan uptake. These phenotypes reflect the role of ergosterol as a regulator of membrane permeability and fluidity. Genetic mapping experiments revealed that ERG6 is located on chromosome XIII, tightly linked to sec59.     

Phosphatidyl ethanolamine is essential for targeting the arginine transporter Can1p to the plasma membrane of yeast

In continuation of our previous study, we show that phosphatidyl ethanolamine (PE) depletion affects, in addition to amino acid transporters, activities of at least two other proton motive force (pmf)-driven transporters (Ura4p and Mal6p). For Can1p, we demonstrate that the lack of PE results in a failure of the permease targeting to plasma membrane. Despite the pleiotropic effect of PE depletion, a specific role of PE in secretion of a defined group of permeases can be distinguished. Pmf-driven transporters are more sensitive to the lack of PE than other plasma membrane proteins.     

C-terminal domain of APETALA1 is essential for its functional divergence from CAULIFLOWER in Arabidopsis

Journal of Plant Biochemistry and Biotechnology - APETALA1 (AP1) and CAULIFLOWER (CAL) are involved in floral meristem identity and suppress the inflorescence meristem program in flower meristem in...     

The C-terminal sequence of LMADS1 is essential for the formation of homodimers for B function proteins

LMADS1, a lily (Lilium longiflorum) AP3 orthologue, contains the complete consensus sequence of the paleoAP3 (YGSHDLRLA) and PI-derived (YEFRVQPSQPNLH) motifs in the C-terminal region of the protein. Interestingly, through yeast two-hybrid analysis, LMADS1 was found to be capable of forming homodimers. These results indicated that LMADS1 represents an ancestral form of the B function protein, which retains the ability to form homodimers in regulating petal and stamen development in lily. To explore the involvement of the conserved motifs in the C-terminal region of LMADS1 in forming homodimers, truncated forms of LMADS1 were generated, and their ability to form homodimers was analyzed using yeast two-hybrid and electrophoretic mobility shift assay. The ability of LMADS1 to form homodimers decreased once the C-terminal paleoAP3 motif was deleted. When both paleoAP3 and PI-derived motifs were deleted, the ability of LMADS1 to form homodimers was completely abolished. This result indicated that although the paleoAP3 motif promotes the formation of LMADS1 homodimers, the PI-derived motif is essential. Deletion analysis indicated that two amino acids, RV, of the 5 final amino acids, YEFRV, in the PI-derived motif are essential for the formation of homodimers. Further, point mutation analysis indicated that amino acid Val was absolutely necessary, whereas residue Arg played a less important role in the formation of homodimers. Furthermore, Arabidopsis AP3 was able to form homodimers once its C-terminal region was replaced by that of LMADS1. This result indicated that the C-terminal region of LMADS1 is responsible and essential for homodimer formation of the ancestral form of the B function protein.