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Intracerebral aneurysms (IAs) are pathological dilatations of cerebral arteries whose rupture leads to subarachnoid hemorrhage, a significant cause of disability and death. Inflammation is recognized as a critical contributor to the formation, growth, and rupture of IAs; however, its precise actors have not yet been fully elucidated. Here, we report CNS-associated macrophages (CAMs), also known as border-associated macrophages, as one of the key players in IA pathogenesis, acting as critical mediators of inflammatory processes related to IA ruptures. Using a new mouse model of middle cerebral artery (MCA) aneurysms we show that CAMs accumulate in the IA walls. This finding was confirmed in a human MCA aneurysm obtained after surgical clipping, together with other pathological characteristics found in the experimental model including morphological changes and inflammatory cell infiltration. In addition, in vivo longitudinal molecular MRI studies revealed vascular inflammation strongly associated with the aneurysm area, i.e., high expression of VCAM-1 and P-selectin adhesion molecules, which precedes and predicts the bleeding extent in the case of IA rupture. Specific CAM depletion by intracerebroventricular injection of clodronate liposomes prior to IA induction reduced IA formation and rupture rate. Moreover, the absence of CAMs ameliorated the outcome severity of IA ruptures resulting in smaller hemorrhages, accompanied by reduced neutrophil infiltration. Our data shed light on the unexplored role of CAMs as main actors orchestrating the progression of IAs towards a rupture-prone state., (© 2024. The Author(s).)

Background: Sporadic venous malformation (VM) and angiomatosis of soft tissue (AST) are benign, congenital vascular anomalies affecting venous vasculature. Depending on the size and location of the lesion, symptoms vary from motility disturbances to pain and disfigurement. Due to the high recurrence of the lesions, more effective therapies are needed., Methods: As targeting stromal cells has been an emerging concept in anti-angiogenic therapies, here, by using VM/AST patient samples, RNA-sequencing, cell culture techniques, and a xenograft mouse model, we investigated the crosstalk of endothelial cells (EC) and fibroblasts and its effect on vascular lesion growth., Results: We report, for the first time, the expression and secretion of transforming growth factor A (TGFA) in ECs or intervascular stromal cells in AST and VM lesions. TGFA induced secretion of vascular endothelial growth factor (VEGF-A) in paracrine fashion, and regulated EC proliferation. Oncogenic PIK3CA variant in p.H1047R, a common somatic mutation found in these lesions, increased TGFA expression, enrichment of hallmark hypoxia, and in a mouse xenograft model, lesion size, and vascularization. Treatment with afatinib, a pan-ErbB tyrosine-kinase inhibitor, decreased vascularization and lesion size in a mouse xenograft model with ECs expressing oncogenic PIK3CA p.H1047R variant and fibroblasts., Conclusions: Based on the data, we suggest that targeting of both intervascular stromal cells and ECs is a potential treatment strategy for vascular lesions having a fibrous component., Funding: Academy of Finland, Ella and Georg Ehnrooth foundation, the ERC grants, Sigrid Jusélius Foundation, Finnish Foundation for Cardiovascular Research, Jane and Aatos Erkko Foundation, GeneCellNano Flagship program, and Department of Musculoskeletal and Plastic Surgery, Helsinki University Hospital., Competing Interests: SJ, HI, PV, HR, HP, SK, MK, JL, NL, SL, MB, EA, KL, JL, JA, TÖ, MK, PS, ET, SY, JL No competing interests declared, (© 2023, Jauhiainen, Ilmonen et al.)

Background: Brain arteriovenous malformations (bAVM) may rupture causing disability or death. BAVM vessels are characterized by abnormally high flow that in general triggers expansive vessel remodeling mediated by cyclo-oxygenase-2 (COX2), the target of non-steroidal anti-inflammatory drugs. We investigated whether COX2 is expressed in bAVMs and whether it associates with inflammation and haemorrhage in these lesions., Methods: Tissue was obtained from surgery of 139 bAVMs and 21 normal Circle of Willis samples. The samples were studied with immunohistochemistry and real-time quantitative polymerase chain reaction (RT-PCR). Clinical data was collected from patient records., Results: COX2 expression was found in 78% (109/139) of the bAVMs and localized to the vessels' lumen or medial layer in 70% (95/135) of the bAVMs. Receptors for prostaglandin E2, a COX2-derived mediator of vascular remodeling, were found in the endothelial and smooth muscle cells and perivascular inflammatory cells of bAVMs. COX2 was expressed by infiltrating inflammatory cells and correlated with the extent of inflammation (r = .231, p = .007, Spearman rank correlation). COX2 expression did not associate with haemorrhage., Conclusion: COX2 is induced in bAVMs, and possibly participates in the regulation of vessel wall remodelling and ongoing inflammation. Role of COX2 signalling in the pathobiology and clinical course of bAVMs merits further studies., (© 2021. The Author(s).)

Background and Purpose: Periodontal infections are associated with the formation and rupture of intracranial aneurysms (IAs). This study investigated the role of two key periodontal pathogens, Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans., Methods: Immunoglobulin A (IgA) and IgG antibodies against P. gingivalis and A. actinomycetemcomitans were measured with enzyme immune assay from the serum of 227 IA patients, of whom 64 also underwent clinical oral examination. As a control group, 1096 participants in a cross-sectional health survey, Health 2000, underwent serological studies and oral examination. Logistic regression was used for multivariate analysis. Immunohistochemistry was performed to demonstrate bacteria-derived epitopes in the IA wall., Results: Widespread gingivitis and severe periodontitis were more common in IA patients than in controls (2× and 1.5×, respectively). IgA antibodies against P. gingivalis and A. actinomycetemcomitans were 1.5× and 3-3.4× higher, respectively, in both unruptured and ruptured IA patients compared to controls (p ≤ 0.003). IgG antibodies against P. gingivalis were 1.8× lower in unruptured IA patients (p < 0.001). In multivariate analysis, high IgA, but low IgG, antibody levels against P. gingivalis (odds ratio [OR] = 1.4, 95% confidence interval [Cl] = 1.1-1.8 and OR = 1.5, 95% Cl = 1.1-1.9; OR = 0.6, 95% Cl = 0.4-0.7 and OR = 0.5, 95% Cl = 0.4-0.7) and against A. actinomycetemcomitans (OR = 2.3, 95% Cl = 1.7-3.1 and OR = 2.1, 95% Cl = 1.5-2.9; OR = 0.6, 95% Cl = 0.4-0.8 and OR = 0.6, 95% Cl = 0.5-0.9) were associated with the risk of IA formation and rupture. Immunohistochemistry showed P. gingivalis epitopes in the IA wall., Conclusions: Exposure to the periodontal pathogens P. gingivalis and A. actinomycetemcomitans and dysfunctional acquired immune response against them may increase the risk of IA formation and IA rupture., (© 2021 The Authors. European Journal of Neurology published by John Wiley & Sons Ltd on behalf of European Academy of Neurology.)

Degeneration of intracranial aneurysm wall is under active research and recent studies indicate an increased risk of rupture of intracranial aneurysm among patients with periodontal diseases. In addition, oral bacterial DNA has been identified from wall samples of ruptured and unruptured aneurysms. These novel findings led us to evaluate if oral diseases could predispose to pathological changes seen on intracranial aneurysm walls eventually leading to subarachnoid hemorrhage. The aim of this review is to consider mechanisms on the relationship between periodontitis and aneurysm rupture, focusing on recent evidence.

Background: Arteriovenous malformations of the brain (bAVM) may rupture from aneurysms or ectasias of the feeding, draining, or nidal vessels. Moreover, they may rupture from the immature, fragile nidal vessels that are characteristic to bAVMs. How the histopathological changes of the nidal vessels associate with clinical presentation and hemorrhage of the lesion is not well known., Materials and Methods: We investigated tissue samples from surgically treated bAVMs (n = 85) using standard histological and immunohistochemical stainings. Histological features were compared with the clinical presentation of the patient., Results: Microhemorrhages from nidal vessels were found both in bAVMs with a history of clinically evident rupture and in bAVMs considered unruptured. These microhemorrhages were associated with presence of immature, pathological nidal vessels (p = 0.010) and perivascular inflammation of these vessels (p = 0.001), especially with adhesion of neutrophils (p < 0.001). In multivariate analysis, perivascular inflammation (OR = 19, 95% CI 1.6 to 230), neutrophil infiltration of the vessel wall (OR = 13, 95% CI 1.9 to 94), and rupture status (OR = 0.13, 95% CI 0.017 to 0.92) were significantly associated with microhemorrhages., Conclusions: Clinically silent microhemorrhages from nidal vessels seem to be very common in bAVMs, and associate with perivascular inflammation and neutrophil infiltration. Further studies on the role of perivascular inflammation in the clinical course of bAVMs are indicated.

Background: Hemorrhage from an arteriovenous malformation of the brain (bAVM) has been associated with focal inflammation of the bAVM. Intrigued by the possibility of anti-inflammatory drug therapy to stabilize bAVMs and prevent hemorrhage, we investigated the association of bAVM inflammation with other histological features and clinical presentation., Materials and Methods: Tissue samples from 85 surgically treated bAVMs were studied with histology and CD45 immunostainings. The histological data was compared with the clinical history of the patient. Univariate analysis and logistic regression were performed., Results: Inflammation was found in all studied bAVMs and did not associate with rupture (p = 0.442). While multiple types of inflammatory cells were present, macrophages were clearly the dominant inflammatory cell type, especially in samples with strong inflammation (87% of the samples). Of those bAVMs that had strong inflammation, only 56% had presented with clinically evident rupture. However, hemosiderin which is a sign of prior hemorrhage was detected in 78.4% (58/74) of samples with strong inflammation and was associated with it (p = 0.003). Inflammation in the nidus and parenchyma was associated with perivascular inflammation (p < 0.001). Multivariate analysis did not reveal any independent histological or clinical risk factor for inflammation., Conclusions: Since strong inflammation is present in both unruptured and ruptured bAVMs, it is not just a reaction to rupture. Our observations suggest that inflammation of the bAVM may indeed predispose to fragility and hemorrhage of the nidal vessels. Further studies in the role of inflammation in the untreated clinical course of bAVMs are indicated.

Purpose: The aim of this study was to study the effect of the embryo freezing method on the birth weight of newborns from frozen embryo transfer (FET) cycles, and the pregnancy results of cleavage stage embryos cryopreserved by slow freezing or vitrification., Methods: This is a retrospective cohort study undertaken in a University Hospital IVF unit using concurrently both the slow-freezing and the vitrification techniques. All frozen-thawed and vitrified-warmed day 2 and day 3 embryo transfers during the time period from 1 April 2009 to 31 November 2013 were included in the study., Results: There was no statistically significant weight difference between newborns from vitrified or slow-frozen embryos (3588 vs 3670 g). A higher post-thaw viability rate was achieved after cryopreservation by the vitrification technique compared to the slow-freezing protocol (83.4 vs 61.4%). The miscarriage rate was lower in the vitrification group (15.7 vs 29.0%). The live birth rates were similar (19.5 vs 19.1%) in the slow-freezing and vitrification groups, respectively. Among vitrified embryos, 7.4 embryos needed to be thawed to produce one delivery; in the slow-freezing group, that number was 11.9., Conclusions: The freezing method has no impact on the weight of the newborn. With lower post-thaw survival rates and higher miscarriage rates, the slow-freezing cryopreservation protocol is inferior to the vitrification technique.

There are more firearms in Northern Finland as compared to Southern Finland, and a positive association between suicide rates and the number of firearms in a given region has been demonstrated in previous literature. Accordingly, the authors compared firearm suicide rates of Finnish adolescent (under 18 years) males in the two geographic regions. Young adult (18-24 years) and adult (25-44 years) males were used as reference groups. National data on cases of suicide in Northern and Southern Finland between 1972 and 2009 were obtained from Statistics Finland. Firearm suicides (n=5,423) were extracted according to ICD-classification (ICD-8/9: E955, ICD-10: X72-X75). The distribution of types of firearms (hunting gun, handgun, other) employed in suicides was also investigated. The adolescent male firearm suicide rate in Northern Finland was almost three times higher than that observed in Southern Finland, while there was no difference in rates of suicide by other methods. A northern excess in firearm suicide rates was also found among young adult and adult males. Hunting guns were the most common type of firearms employed in young male suicides, and their use was especially common in Northern Finland. Our results indicate that the use of firearms plays a major role in explaining the northern excess in young Finnish male suicide rates, and emphasize a need to advance suicide prevention according to specific regional characteristics.

Background: There are only a few recent studies on secular trends in child and adolescent suicides. We examine here trends in rates and methods of suicide among young people in Finland, where suicide rates at these ages are among the highest in the world., Methods: The data, obtained from Statistics Finland, consisted of all suicides (n = 901) committed by persons under 18 years of age over the period 1969-2008. Gender-specific trends were analysed separately for the years 1969-1989 and 1990-2008 using 3-year moving averages. Trends in methods of suicide were examined from 1975 to 2008 in five-year periods., Results: The male-to-female ratio in youth suicides was 3.6:1. The male rates increased in 1969-1989, while the rates among females were inconsistent. After 1990, the rates decreased for males but turned to an increase among females. Shooting was the most common suicide method among males throughout the period, while hanging exceeded poisoning as the most common method among females after 1990. All violent suicides decreased for males and increased for females in 1990-2008., Conclusions: The increase in violent, i.e., more lethal, suicide methods among young females is alarming, as females are known to have higher rates of attempted suicide than males. Alcohol consumption, rates and treatment of depression and violent behaviour among adolescents are discussed as approaches towards explaining this phenomenon., (© 2011 The Authors. Journal of Child Psychology and Psychiatry © 2011 Association for Child and Adolescent Mental Health.)

The Sec61 protein translocation complex in the endoplasmic reticulum (ER) membrane is composed of three subunits. The alpha-subunit, called Sec61p in yeast, is a multispanning membrane protein that forms the protein conducting channel. The functions of the smaller, carboxyl-terminally tail-anchored beta subunit Sbh1p, its close homologue Sbh2p, and the gamma subunit Sss1p are not well understood. Here we show that co-translational protein translocation into the ER is reduced in sbh1Delta sbh2Delta cells, whereas there is a limited reduction of post-translational translocation and no effect on export of a mutant form of alpha-factor precursor for ER-associated degradation in the cytosol. The translocation defect and the temperature-sensitive growth phenotype of sbh1Delta sbh2Delta cells were rescued by expression of the transmembrane domain of Sbh1p alone, and the Sbh1p transmembrane domain was sufficient for coimmunoprecipitation with Sec61p and Sss1p. Furthermore, we show that Sbh1p co-precipitates with the ER transmembrane protein Rtn1p. Sbh1p-Rtn1p complexes do not appear to contain Sss1p and Sec61p. Our results define the transmembrane domain as the minimal functional domain of the Sec61beta homologue Sbh1p in ER translocation, identify a novel interaction partner for Shb1p, and imply that Sbh1p has additional functions that are not directly linked to protein translocation in association with the Sec61 complex.

We identified in a yeast two-hybrid screen the EF-hand Ca(2+)-binding protein Cab45 as an interaction partner of Munc18b. Although the full-length Cab45 resides in Golgi lumen, we characterize a cytosolic splice variant, Cab45b, expressed in pancreatic acini. Cab45b is shown to bind (45)Ca(2+), and, of its three EF-hand motifs, EF-hand 2 is demonstrated to be crucial for the ion binding. Cab45b is shown to interact with Munc18b in an in vitro assay, and this interaction is enhanced in the presence of Ca(2+). In this assay, Cab45b also binds the Munc18a isoform in a Ca(2+)-dependent manner. The endogenous Cab45b in rat acini coimmunoprecipitates with Munc18b, syntaxin 2, and syntaxin 3, soluble N-ethylmaleimide-sensitive factor attachment protein receptors with key roles in the Ca(2+)-triggered zymogen secretion. Furthermore, we show that Munc18b bound to syntaxin 3 recruits Cab45b onto the plasma membrane. Importantly, antibodies against Cab45b are shown to inhibit in a specific and dose-dependent manner the Ca(2+)-induced amylase release from streptolysin-O-permeabilized acini. The present study identifies Cab45b as a novel protein factor involved in the exocytosis of zymogens by pancreatic acini.

We have sequenced the nonstructural protein coding region of Semliki Forest virus temperature-sensitive (ts) mutant strains ts1, ts6, ts9, ts10, ts11, ts13, and ts14. In each case, the individual amino acid changes uncovered were transferred to the prototype strain background and thereby identified as the underlying cause of the altered RNA synthesis phenotype. All mutations mapping to the protease domain of nonstructural protein nsP2 caused defects in nonstructural polyprotein processing and subgenomic RNA synthesis, and all mutations in the helicase domain of nsP2 affected subgenomic RNA production. These types of defects were not associated with mutations in other nonstructural proteins.

In this study, we have analyzed the association of the Sec1p interacting protein Mso1p with the membrane fusion machinery in yeast. We show that Mso1p is essential for vesicle fusion during prospore membrane formation. Green fluorescent protein-tagged Mso1p localizes to the sites of exocytosis and at the site of prospore membrane formation. In vivo and in vitro experiments identified a short amino-terminal sequence in Mso1p that mediates its interaction with Sec1p and is needed for vesicle fusion. A point mutation, T47A, within the Sec1p-binding domain abolishes Mso1p functionality in vivo, and mso1T47A mutant cells display specific genetic interactions with sec1 mutants. Mso1p coimmunoprecipitates with Sec1p, Sso1/2p, Snc1/2p, Sec9p, and the exocyst complex subunit Sec15p. In sec4-8 and SEC4I133 mutant cells, association of Mso1p with Sso1/2p, Snc1/2p, and Sec9p is affected, whereas interaction with Sec1p persists. Furthermore, in SEC4I133 cells the dominant negative Sec4I133p coimmunoprecipitates with Mso1p-Sec1p complex. Finally, we identify Mso1p as a homologue of the PTB binding domain of the mammalian Sec1p binding Mint proteins. These results position Mso1p in the interface of the exocyst complex, Sec4p, and the SNARE machinery, and reveal a novel layer of molecular conservation in the exocytosis machinery.

We report here identification and characterization of a mutation in the GPI14 gene, the yeast homologue of the mammalian PIG-M that functions in the synthesis of the GPI moiety anchoring proteins to the plasma membrane. We show that the first putative transmembrane domain of Gpi14p is not essential for its function. Downregulation of GPI14 expression/reduced protein function due to an amino terminal deletion resulted in increased transcription and production of an endogenous and a heterologous secreted protein expressed from HSP150 and ADH1 promoter, respectively. In these cells, unfolded protein response was induced but was not responsible for the enhanced production of these proteins. A cell wall defect in the gpi14 mutant cells was suggested by cell aggregation phenotype, increased sensitivity to Calcofluor white, an increased release of Gas1p and total protein into the culture medium. In the gpi14 mutant cells, transcription of RLM1, a transcription factor participating in the cell wall integrity signalling pathway, was increased, and deletion of RLM1 resulted in a synthetic lethal phenotype with the gpi14 mutation. These results suggest that partial inactivation of Gpi14p causes defects in the cell wall structure and suggest that compromised GPI anchor synthesis results in enhanced protein production via the cell wall integrity signalling pathway., (Copyright 2005 John Wiley & Sons, Ltd.)

The SEB1/SBH1 and the SSO genes encode components of the protein secretory machinery functioning at the opposite ends, ER translocation and exocytosis, respectively, of the secretory pathway in Saccharomyces cerevisiae. Overexpression of these genes can rescue temperature-sensitive (ts) growth defect of many sec mutants impaired in protein secretion. Furthermore, their overexpression in wild-type yeast enhances production of secreted proteins in S. cerevisiae, which suggests that they may be rate-limiting factors in this process. Here we report isolation of Kluyveromyces lactis homologues of these genes. KlSSO1 and KlSEB1 were isolated as clones capable of rescuing growth of ts sso2-1 and seb1Delta seb2Delta sem1Delta strains, respectively, at the restrictive temperature. The encoded Kluyveromyces proteins are up to 70% identical with the S. cerevisiae homologues at the amino acid level and can functionally replace them. Interestingly, KlSSO1 and KlSEB1 show similar enhancing effect on production of a secreted protein as the SSO and SEB1 genes of S. cerevisiae when overexpressed. In accordance with the high homology level of the secretory pathway proteins in different yeast species, the polyclonal antibodies raised against S. cerevisiae Seb1p, Sso2p and Sec4p can detect homologous proteins in cell lysates of K. lactis and Pichia pastoris, the latter also in Candida utilis. The GenBank Accession Nos are AF307983 (K. lactis SSO1) and AF318314 (K. lactis SEB1)., (Copyright (c) 2004 John Wiley & Sons, Ltd.)

We describe here a screening procedure devised for searching new genes involved in protein secretion in Saccharomyces cerevisiae. The screening procedure takes advantage of yeast strains constructed within the EUROFAN project, in which the promoters of the novel essential genes were replaced by the doxycycline-regulated tetO(7)-CYC1 promoter. This promoter is active in normal growth medium but results in downregulation of the gene in the presence of doxycycline. The yeast cells were grown in the presence or absence of doxycycline, and both the growth and secretion of the heat shock protein, Hsp150p, into the culture medium were determined. In seven strains there was a specific effect on protein secretion. In a strain in which the RPN5 gene was downregulated, the level of secreted Hsp150p was increased compared to the control culture. When RER2 was downregulated, cells secreted Hsp150p that was not of the mature size. In five strains, secretion was more severely reduced than cell growth. One of these downregulated genes, YGL098w, was recently reported to encode an ER-located t-SNARE, USE1. Four of the genes detected, NOG2, NOP15, RRP40 and SDA1, encode proteins involved in ribosome assembly, suggesting a possible new signalling pathway between ribosome biogenesis and production of secreted proteins. The results obtained here indicate that the present screen could be successfully used in larger scale to identify novel secretion-related genes., (Copyright 2004 John Wiley & Sons, Ltd.)

The yeast Saccharomyces cerevisiae has two closely related plasma membrane syntaxins, Sso1p and Sso2p, which together provide an essential function in vegetative cells. However, Sso1p is also specifically needed during sporulation; and this function cannot be provided by Sso2p. We used fusions between SSO1 and SSO2 to map the sporulation-specific function of SSO1. We found that the two N-terminal alpha-helices Ha and Hb of Sso1p are important for sporulation, since it is reduced 8-fold for fusions where Ha and Hb are derived from Sso2p. In contrast, the C-terminal half of Sso1p does not seem to be specifically required for sporulation. Surprisingly, we further found that the 3' untranslated region (3'UTR) of SSO1 is essential for sporulation. Western blots failed to reveal a preferential expression of Sso1p in sporulating cells, indicating that effects on gene expression are unlikely to explain why the SSO1 3'UTR is needed for sporulation.

Highly polarized exocytosis of vesicles at hyphal apices is an essential requirement of tip growth. This requirement may be met by the localization and/or activation of an apical SNARE-based machinery. We have cloned nsyn1 and nsyn2, SNAREs predicted to function at the plasma membrane in Neurospora crassa. Transformation of extra copies of nsyn1 into wild-type strains displayed effects consistent with quelling of nsyn1 expression, which was lethal in most transformants. All surviving transformants grew slowly, conidiated poorly, and were male sterile. In addition, antisense nsyn1 strains grew slowly, with abnormal hyphal diameters and polarity and defective conidiation. For nsyn2, several repeat induced point mutation (RIP) crosses produced no, or poorly germinating ascospores. Those that germinated produced slow-growing hyphae with abnormal branching. The defects in nsyn1 and nsyn2 mutants are consistent with differential impaired vesicle fusion in hyphal tips and other developmental stages.

Intrabody technology was applied to characterize the function and intracellular localization of a highly conserved Saccharomyces cerevisiae Sem1 protein. DSS1, the mammalian homologue of Sem1p, is functionally conserved between yeast and mammalian cells, and in mammalian cells physically interacts with the strong tumour supressor BRCA2. Yeast and the generated intrabodies are thus expected to offer a useful system for studies on Sem1p/DSS1 function. Sem1p-specific antibody isolated from a phage display library was expressed intracellularily and targeted to either the cytosol or the nucleus of yeast cells. Analysis of the applicability of different antibody fragments as intrabodies showed that the Fab intrabody was expressed most efficiently. Expression of nuclear-targeted anti-Sem1p Fab intrabodies inhibited the growth of the sigma1278b yeast strain in a manner similar to deletion of the SEM1 gene. This indicates that the Fab intrabodies interact in vivo with Sem1p and result in inactivation of Sem1p. Localization of the Fab intrabody with or without the nuclear localization signal to the nucleus in Sem1p-dependent manner suggests that Sem1p mediates the nuclear transport of the intrabody without any targeting signal. Our results suggest that Sem1p function in yeast cells is in part manifested in the nucleus., (Copyright 2003 John Wiley & Sons, Ltd.)

The exocyst is a conserved protein complex proposed to mediate vesicle tethering at the plasma membrane. Previously, we identified SEB1/SBH1, encoding the beta subunit of the Sec61p ER translocation complex, as a multicopy suppressor of the sec15-1 mutant, defective for one subunit of the exocyst complex. Here we show the functional and physical interaction between components of endoplasmic reticulum translocon and the exocytosis machinery. We show that overexpression of SEB1 suppresses the growth defect in all exocyst sec mutants. In addition, overexpression of SEC61 or SSS1 encoding the other two components of the Sec61p complex suppressed the growth defects of several exocyst mutants. Seb1p was coimmunoprecipitated from yeast cell lysates with Sec15p and Sec8p, components of the exocyst complex, and with Sec4p, a secretory vesicle associated Rab GTPase that binds to Sec15p and is essential for exocytosis. The interaction between Seb1p and Sec15p was abolished in sec15-1 mutant and was restored upon SEB1 overexpression. Furthermore, in wild type cells overexpression of SEB1 as well as SEC4 resulted in increased production of secreted proteins. These findings propose a novel functional and physical link between the endoplasmic reticulum translocation complex and the exocyst.

The 14-3-3 proteins are highly conserved, ubiquitously expressed proteins taking part in numerous cellular processes. Two genes encoding 14-3-3 proteins, ftt1 and ftt2, were isolated and characterised from the filamentous fungus Trichoderma reesei. FTTI showed the highest sequence identity (98% at the amino acid level) to the Trichoderma harzianum protein Th1433. FTTII is relatively distinct from FTTI, showing approximately 75% identity to other fungal 14-3-3 proteins. Despite their sequence divergence, both of the T. reesei ftt genes were equally able to complement the yeast bmh1 bmh2 double disruption. The T. reesei ftt genes were also found to be quite closely linked in the genomic DNA. A C-terminally truncated version of ftt1 (ftt1DeltaC) was first isolated as a multicopy suppressor of the growth defect of the temperature-sensitive yeast secretory mutant sec15-1. Overexpression of ftt1DeltaC also suppressed the growth defect of sec2-41, sec3-101, and sec7-1 strains. Overexpression of ftt1DeltaC in sec2-41 and sec15-1 strains could also rescue the secretion of invertase at the restrictive temperatures, and overexpression of full-length ftt1 enhanced invertase secretion by wild-type yeast cells. These findings strongly suggest that the T. reesei ftt1 has a role in protein secretion.