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N J Bryant

Publications and source records attributed to N J Bryant.

17 recordsLinked to original sources
PubMed2001

Vps45p stabilizes the syntaxin homologue Tlg2p and positively regulates SNARE complex formation.

Sec1p-like/Munc-18 (SM) proteins bind to t-SNAREs and inhibit ternary complex formation. Paradoxically, the absence of SM proteins does not result in constitutive membrane fusion. Here, we show that in yeast cells lacking the SM protein Vps45p, the t-SNARE Tlg2p is down-regulated, to undetectable levels, by rapid proteasomal degradation. In the absence of Vps45p, Tlg2p can be stabilized through abolition of proteasome activity. Surprisingly, the stabilized Tlg2p was targeted to the correct intracellular location. However, the stabilized Tlg2p is non-functional and unable to bind its cognate SNARE binding partners, Tlg1p and Vti1p, in the absence of Vps45p. A truncation mutant lacking the first 230 residues of Tlg2p no longer bound Vps45p but was able to form complexes with Tlg1p and Vti1p in the absence of the SM protein. These data provide us with two valuable insights into the function of SM proteins. First, SM proteins act as chaperone-like molecules for their cognate t-SNAREs. Secondly, SM proteins play an essential role in the activation process allowing their cognate t-SNARE to participate in ternary complex formation.

Carrier Proteins↗
PubMed2001

Syntaxin 7 complexes with mouse Vps10p tail interactor 1b, syntaxin 6, vesicle-associated membrane protein (VAMP)8, and VAMP7 in b16 melanoma cells.

Syntaxin 7 is a mammalian target soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) involved in membrane transport between late endosomes and lysosomes. The aim of the present study was to use immunoaffinity techniques to identify proteins that interact with Syntaxin 7. We reasoned that this would be facilitated by the use of cells producing high levels of Syntaxin 7. Screening of a large number of tissues and cell lines revealed that Syntaxin 7 is expressed at very high levels in B16 melanoma cells. Moreover, the expression of Syntaxin 7 increased in these cells as they underwent melanogenesis. From a large scale Syntaxin 7 immunoprecipitation, we have identified six polypeptides using a combination of electrospray mass spectrometry and immunoblotting. These polypeptides corresponded to Syntaxin 7, Syntaxin 6, mouse Vps10p tail interactor 1b (mVti1b), alpha-synaptosome-associated protein (SNAP), vesicle-associated membrane protein (VAMP)8, VAMP7, and the protein phosphatase 1M regulatory subunit. We also observed partial colocalization between Syntaxin 6 and Syntaxin 7, between Syntaxin 6 and mVti1b, but not between Syntaxin 6 and the early endosomal t-SNARE Syntaxin 13. Based on these and data reported previously, we propose that Syntaxin 7/mVti1b/Syntaxin 6 may form discrete SNARE complexes with either VAMP7 or VAMP8 to regulate fusion events within the late endosomal pathway and that these events may play a critical role in melanogenesis.

Amino Acid Sequence↗
PubMed2001

Balloon cell melanoma in three dogs: a histopathological, immunohistochemical and ultrastructural study.

Balloon cell melanoma, a variant of malignant melanoma, has been reported on rare occasions in animals and is uncommon in man. Such tumours have variable numbers of large, round to polygonal cells with abundant, clear, often vacuolated cytoplasm containing fine melanin granules and variable amounts of lipid. This report describes balloon cell melanomas in three dogs. Immunohistochemically, these tumours showed reactions similar to those of human melanomas when tested with antibodies against S-100 protein, neuron-specific enolase (NSE) and vimentin. Electron microscopically, numerous heterogeneous melanosomes were demonstrated in the balloon cell cytoplasm of one tumour. Although balloon cell melanoma apparently occurs infrequently in dogs, it should always be considered in the differential diagnosis of neoplasms containing clear cells.

Animals↗
PubMed2000

Localization of phosphatidylinositol 3-phosphate in yeast and mammalian cells.

Phosphatidylinositol 3-kinase (PI3K) regulates several vital cellular processes, including signal transduction and membrane trafficking. In order to study the intracellular localization of the PI3K product, phosphatidylinositol 3-phosphate [PI(3)P], we constructed a probe consisting of two PI(3)P-binding FYVE domains. The probe was found to bind specifically, and with high affinity, to PI(3)P both in vitro and in vivo. When expressed in fibroblasts, a tagged probe localized to endosomes, as detected by fluorescence microscopy. Electron microscopy of untransfected fibroblasts showed that PI(3)P is highly enriched on early endosomes and in the internal vesicles of multivesicular endosomes. While yeast cells deficient in PI3K activity (vps15 and vps34 mutants) were not labelled, PI(3)P was found on intralumenal vesicles of endosomes and vacuoles of wild-type yeast. vps27Delta yeast cells, which have impaired endosome to vacuole trafficking, showed a decreased vacuolar labelling and increased endosome labelling. Thus PI(3)P follows a conserved intralumenal degradation pathway, and its generation, accessibility and turnover are likely to play a crucial role in defining the early endosome and the subsequent steps leading to multivesicular endosome formation.

Animals↗
PubMed2000

VPS21 controls entry of endocytosed and biosynthetic proteins into the yeast prevacuolar compartment.

Mutations in the VPS (vacuolar protein sorting) genes of Saccharomyces cerevisiae have been used to define the trafficking steps that soluble vacuolar hydrolases take en route from the late Golgi to the vacuole. The class D VPS genes include VPS21, PEP12, and VPS45, which appear to encode components of a membrane fusion complex involved in Golgi-to-endosome transport. Vps21p is a member of the Rab family of small Ras-like GTPases and shows strong homology to the mammalian Rab5 protein, which is involved in endocytosis and the homotypic fusion of early endosomes. Although Rab5 and Vps21p appear homologous at the sequence level, it has not been clear if the functions of these two Rabs are similar. We find that Vps21p is an endosomal protein that is involved in the delivery of vacuolar and endocytosed proteins to the vacuole. Vacuolar and endocytosed proteins accumulate in distinct transport intermediates in cells that lack Vps21p function. Therefore, it appears that Vps21p is involved in two trafficking steps into the prevacuolar/late endosomal compartment.

Alkaline Phosphatase↗
PubMed1998

Retrograde traffic out of the yeast vacuole to the TGN occurs via the prevacuolar/endosomal compartment.

A large number of trafficking steps occur between the last compartment of the Golgi apparatus (TGN) and the vacuole of the yeast Saccharomyces cerevisiae. To date, two intracellular routes from the TGN to the vacuole have been identified. Carboxypeptidase Y (CPY) travels through a prevacuolar/endosomal compartment (PVC), and subsequently on to the vacuole, while alkaline phosphatase (ALP) bypasses this compartment to reach the same organelle. Proteins resident to the TGN achieve their localization despite a continuous flux of traffic by continually being retrieved from the distal PVC by virtue of an aromatic amino acid-containing sorting motif. In this study we report that a hybrid protein based on ALP and containing this retrieval motif reaches the PVC not by following the CPY sorting pathway, but instead by signal-dependent retrograde transport from the vacuole, an organelle previously thought of as a terminal compartment. In addition, we show that a mutation in VAC7, a gene previously identified as being required for vacuolar inheritance, blocks this trafficking step. Finally we show that Vti1p, a v-SNARE required for the delivery of both CPY and ALP to the vacuole, uses retrograde transport out of the vacuole as part of its normal cellular itinerary.

Alkaline Phosphatase↗
PubMed1998

Traffic into the prevacuolar/endosomal compartment of Saccharomyces cerevisiae: a VPS45-dependent intracellular route and a VPS45-independent, endocytic route.

The vps (vacuolar protein sorting) mutants have been used to dissect and characterize the vacuolar biogenesis pathway in the yeast Saccharomyces cerevisiae. The vps mutants were isolated through their loss of ability to correctly sort the vacuolar hydrolase CPY, which travels from Golgi membranes to the vacuole through a prevacuolar compartment. Over 50 VPS genes have been divided into 6 classes according to vacuolar morphology. Mutations in any one of the class E VPS genes, such as VPS27, lead to an exaggerated form of the prevacuolar compartment. This class E compartment contains endocytosed proteins as well as proteins en route to the vacuole, and is thus taken to represent an intersection point between the endocytic and biosynthetic pathways. Mutations in the class D gene VPS45 can be used to define a second transport intermediate along the vacuolar biogenesis pathway, Golgi-derived transport vesicles carrying vacuolar membrane proteins on their way to the vacuole. Here we demonstrate that the Sec1p-like protein Vps45p is required for the fusion of Golgi-derived vesicles with the prevacuolar compartment indicating that VPS45 functions before VPS27 in the vacuolar biogenesis pathway. In addition, we show that VPS45 function is not required for the delivery of endocytosed proteins to the prevacuolar compartment from the plasma membrane suggesting that the function of Vps45p is restricted to a single vesicular pathway.

Biological Transport↗
PubMed1998

Vacuole biogenesis in Saccharomyces cerevisiae: protein transport pathways to the yeast vacuole.

Delivery of proteins to the vacuole of the yeast Saccharomyces cerevisiae provides an excellent model system in which to study vacuole and lysosome biogenesis and membrane traffic. This organelle receives proteins from a number of different routes, including proteins sorted away from the secretory pathway at the Golgi apparatus and endocytic traffic arising from the plasma membrane. Genetic analysis has revealed at least 60 genes involved in vacuolar protein sorting, numerous components of a novel cytoplasm-to-vacuole transport pathway, and a large number of proteins required for autophagy. Cell biological and biochemical studies have provided important molecular insights into the various protein delivery pathways to the yeast vacuole. This review describes the various pathways to the vacuole and illustrates how they are related to one another in the vacuolar network of S. cerevisiae.

Biological Transport↗
PubMed1997

The membrane protein alkaline phosphatase is delivered to the vacuole by a route that is distinct from the VPS-dependent pathway.

Membrane trafficking intermediates involved in the transport of proteins between the TGN and the lysosome-like vacuole in the yeast Saccharomyces cerevisiae can be accumulated in various vps mutants. Loss of function of Vps45p, an Sec1p-like protein required for the fusion of Golgi-derived transport vesicles with the prevacuolar/endosomal compartment (PVC), results in an accumulation of post-Golgi transport vesicles. Similarly, loss of VPS27 function results in an accumulation of the PVC since this gene is required for traffic out of this compartment. The vacuolar ATPase subunit Vph1p transits to the vacuole in the Golgi-derived transport vesicles, as defined by mutations in VPS45, and through the PVC, as defined by mutations in VPS27. In this study we demonstrate that, whereas VPS45 and VPS27 are required for the vacuolar delivery of several membrane proteins, the vacuolar membrane protein alkaline phosphatase (ALP) reaches its final destination without the function of these two genes. Using a series of ALP derivatives, we find that the information to specify the entry of ALP into this alternative pathway to the vacuole is contained within its cytosolic tail, in the 13 residues adjacent to the transmembrane domain, and loss of this sorting determinant results in a protein that follows the VPS-dependent pathway to the vacuole. Using a combination of immunofluorescence localization and pulse/chase immunoprecipitation analysis, we demonstrate that, in addition to ALP, the vacuolar syntaxin Vam3p also follows this VPS45/27-independent pathway to the vacuole. In addition, the function of Vam3p is required for membrane traffic along the VPS-independent pathway.

Alkaline Phosphatase↗
PubMed1997

Two separate signals act independently to localize a yeast late Golgi membrane protein through a combination of retrieval and retention.

The localization of proteins to late-Golgi membranes (TGN) of Saccharomyces cerevisiae is conferred by targeting motifs containing aromatic residues in the cytosolic domains of these proteins. These signals could act by directing retrieval from a post-Golgi compartment or by preventing exit from the TGN. To investigate the mechanism of localization of yeast TGN proteins, we used the heterologous protein A-ALP (consisting of the cytosolic domain of dipeptidyl aminopeptidase A [DPAP A] fused to the transmembrane and luminal domains of the vacuolar protein alkaline phosphatase [ALP]), which localizes to the yeast TGN. Insertion of the aromatic residue-based TGN localization motif (FXFXD) of DPAP A into the cytosolic domain of ALP results in a protein that resides in the TGN. We demonstrate that the FXFXD motif confers Golgi localization through retrieval from a post-Golgi compartment by detecting a post-Golgi processed form of this protein in the TGN. We present an assay that uncouples retrieval-mediated Golgi localization from static retention-based localization, allowing measurement of the rate at which proteins exit the yeast TGN. We also demonstrate that the cytosolic domain of DPAP A contains additional information, separate from the retrieval motif, that slows exit from the TGN. We propose a model for DPAP A localization that involves two distinct mechanisms: one in which the FXFXD motif directs retrieval from a post-Golgi compartment, and a second that slows the rate at which DPAP A exits the TGN.

Alkaline Phosphatase↗
PubMed1996

Human posterior cricoarytenoid muscle compartments. Anatomy and mechanics.

OBJECTIVE: To document the presence and functional significance of distinct anatomical compartments in the human posterior cricoarytenoid muscle (PCA). DESIGN: Anatomic study of human cadaver larynges. SUBJECTS: Seventeen fresh larynges, harvested at autopsy from 8 men and 9 women, with no history of laryngeal disease or surgery. INTERVENTIONS: Twenty-three PCA muscles from 12 human cadaver larynges were dissected. Computed tomographic scanning and rigid body mechanical analysis were used to compute 3-dimensional motion with simulated individual contraction of PCA compartments in 5 fresh larynges. RESULTS: Discrete medial and lateral bellies with different orientations of muscle fibers were found in every muscle. The 2 bellies insert on opposing aspects of the muscular process of the arytenoid. Very little linear translation was effected by either muscle. The axes of rotation attributable to the 2 bellies differed significantly, with the medial belly effecting rotation about a more vertical axis. The axis of rotation for the lateral muscle belly was nearer the anterior posterior axis than that of the medial belly. CONCLUSIONS: These data indicate that PCA muscle contraction results in arytenoid rotation about a variable oblique axis without significant lateral gliding. There are 2 bellies within the human PCA muscle with differing mechanical actions on the cricoarytenoid joint.

Aged↗
PubMed1996

MRI evaluation of vocal fold paralysis before and after type I thyroplasty.

Magnetic resonance imaging (MRI) is a useful modality for three-dimensional evaluation of laryngeal anatomy. The authors present data on patients with unilateral vocal fold paralysis both before and after type I thyroplasty for medialization. Videoendoscopic and acoustic measures were obtained. MRI was performed preoperatively and postoperatively. Three-dimensional descriptive parameters were established to assess implant location. As well, vocal and endoscopic assessment were used to determine a successful result. Magnetic resonance images of two patients with satisfactory results were compared with images and vocal analysis of two dissatisfied patients. Use of MRI after type I thyroplasty provides additional information about implant location that can help determine the cause of poor results.

Aged↗
PubMed1996

The newly identified yeast GRD genes are required for retention of late-Golgi membrane proteins.

Processing of A-ALP, a late-Golgi membrane protein constructed by fusing the cytosolic domain of dipeptidyl aminopeptidase A to the transmembrane and lumenal domains of alkaline phosphatase (ALP), serves as a convenient assay for loss of retention of late-Golgi membrane proteins in Saccharomyces cerevisiae. In this study, a large group of novel grd (for Golgi retention defective) yeast mutants, representing 18 complementation groups, were identified on the basis of their mislocalization of A-ALP to the vacuole, where it was proteolytically processed and thus became enzymatically activated. All of the grd mutants exhibited significant mislocalization of A-ALP, as measured by determining the kinetics of A-ALP processing and by analyzing its

Alkaline Phosphatase↗
PubMed1995

Localization of a protein A-tagged Kex2 protein to the vacuole of Saccharomyces cerevisiae allows rapid purification of vacuolar membranes.

We have previously reported an immunoisolation procedure which allows purification of Kex2p-containing Golgi membranes from lysed yeast cells. In order to evaluate the use of tagging procedures in organelle isolation we set out to isolate the same Golgi membrane fraction using a version of the Kex2 protease that had been affinity-tagged at its C-terminus. This protein is found to be localized in the vacuole, providing the basis of a method for the affinity-purification of vacuolar membranes.

Base Sequence↗
PubMed1993

Immunoisolation of Kex2p-containing organelles from yeast demonstrates colocalisation of three processing proteinases to a single Golgi compartment.

One of the Golgi compartments of Saccharomyces cerevisiae is defined by the presence of a specific endoproteinase, Kex2p, which cleaves precursor polypeptides at pairs of basic residues. We have used antibodies directed against the cytoplasmically disposed C-terminal domain of Kex2p to develop an immuno-affinity procedure for the isolation of Kex2p-containing organelles. The method gives a high yield of sealed organelles that are essentially free of contamination from other secretory pathway organelles while being significantly enriched for two other late Golgi enzymes, dipeptidylaminopeptidase A and the Kex1 carboxypeptidase. Our findings provide clear evidence for a single yeast Golgi compartment containing all three late-processing enzymes, which is likely to be the functional equivalent in yeast of the mammalian trans-Golgi network.

Carboxypeptidases↗
PubMedDate not supplied

A rapid tube technique for fetal cell typing.

A modification of Greendyke technique for fetal cell typing is described in which incubation time and quantities of antisera are reduced without affecting the sensitivity of the test.

Blood Group Antigens↗