scholarly journals Immunolocalization of glyceraldehyde-3-phosphate dehydrogenase, hexokinase, and carboxypeptidase Y in yeast cells at the ultrastructural level.

1987 ◽  
Vol 35 (3) ◽  
pp. 327-333 ◽  
Author(s):  
E van Tuinen ◽  
H Riezman
Keyword(s):  
Immunoelectron Microscopy ◽  
Ultrastructural Level ◽  
Subcellular Fractionation ◽  
Glycolytic Enzymes ◽  
Yeast Cells ◽  
Carboxypeptidase Y ◽  
Yeast Vacuole

We have developed a simple and effective method to embed whole yeast cells in Lowicryl resins with excellent ultrastructural and antigenic preservation. Using affinity-purified antibodies eluted from electrophoretically separated proteins transferred to nitrocellulose, we have shown by immunoelectron microscopy that two glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase and hexokinase, are present in the cytoplasm and the nucleus. Carboxypeptidase Y is localized in the yeast vacuole. These results agree with earlier localization studies based on subcellular fractionation.

scholarly journals Characterization of TPM1 disrupted yeast cells indicates an involvement of tropomyosin in directed vesicular transport.

1992 ◽  
Vol 118 (2) ◽  
pp. 285-299 ◽  
Author(s):  
H Liu ◽  
A Bretscher
Keyword(s):  
Cell Surface ◽  
Secretory Pathway ◽  
Synthetic Lethality ◽  
Vesicular Transport ◽  
Yeast Cells ◽  
Carboxypeptidase Y ◽  
Apparent Loss ◽  
Abnormal Accumulation ◽  
Late Step ◽  
Actin Cables

Disruption of the yeast tropomyosin gene TPM1 results in the apparent loss of actin cables from the cytoskeleton (Liu, H., and A. Bretscher. 1989. Cell. 57:233-242). Here we show that TPM1 disrupted cells grow slowly, show heterogeneity in cell size, have delocalized deposition of chitin, and mate poorly because of defects in both shmooing and cell fusion. The transit time of alpha-factor induced a-agglutinin secretion to the cell surface is longer than in isogenic wild-type strains, and some of the protein is mislocalized. Many of the TPM1-deleted cells contain abundant vesicles, similar in morphology to late secretory vesicles, but without an abnormal accumulation of intermediates in the delivery of either carboxypeptidase Y to the vacuole or invertase to the cell surface. Combinations of the TPM1 disruption with sec13 or sec18 mutations, which affect early steps in the secretory pathway, block vesicle accumulation, while combinations with sec1, sec4 or sec6 mutations, which affect a late step in the secretory pathway, have no effect on the vesicle accumulation. The phenotype of the TPM1 disrupted cells is very similar to that of a conditional mutation in the MYO2 gene, which encodes a myosin-like protein (Johnston, G. C., J. A. Prendergast, and R. A. Singer. 1991. J. Cell Biol. 113:539-551). The myo2-66 conditional mutation shows synthetic lethality with the TPM1 disruption, indicating that the MYO2 and TPM1 gene products may be involved in the same, or parallel function. We conclude that tropomyosin, and by inference actin cables, may facilitate directed vesicular transport of components to the correct location on the cell surface.

The yeast VPS5/GRD2 gene encodes a sorting nexin-1-like protein required for localizing membrane proteins to the late Golgi

1997 ◽  
Vol 110 (9) ◽  
pp. 1063-1072 ◽  
Author(s):  
S.F. Nothwehr ◽  
A.E. Hindes
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Localization ◽  
Endocytic Pathway ◽  
Yeast Cells ◽  
Carboxypeptidase Y ◽  
Golgi Membrane ◽  
Sorting Nexin ◽  
Vacuolar Protein ◽  
Sorting Nexin 1

Genetic analysis of late Golgi membrane protein localization in Saccharomyces cerevisiae has uncovered a large number of genes (called GRD) that are required for retention of A-ALP, a model late Golgi membrane protein. Here we describe one of the GRD genes, VPSS/GRD2, that encodes a hydrophilic protein similar to human sorting nexin-1, a protein involved in trafficking of the epidermal growth factor receptor. In yeast cells containing a vps5 null mutation the late Golgi membrane proteins A-ALP and Kex2p were rapidly mislocalized to the vacuolar membrane. A-ALP was delivered to the vacuole in vps5 mutants in a manner independent of a block in the early endocytic pathway. vps5 null mutants also exhibited defects in both vacuolar morphology and in sorting of a soluble vacuolar protein, carboxypeptidase Y. The latter defect is apparently due to an inability to localize the carboxypeptidase Y sorting receptor, Vps10p, to the Golgi since it is rapidly degraded in the vacuole in vps5 mutants. Fractionation studies indicate that Vps5p is distributed between a free cytosolic pool and a particulate fraction containing Golgi, transport vesicles, and possibly endosomes, but lacking vacuolar membranes. Immunofluorescence microscopy experiments show that the membrane-associated pool of Vps5p localizes to an endosome-like organelle that accumulates in the class E vps27 mutant. These results support a model in which Vps5p is required for retrieval of membrane proteins from a prevacuolar/late endosomal compartment back to the late Golgi apparatus.

GENETIC PROPERTIES OF MUTATIONS AT THE PEP4 LOCUS IN SACCHAROMYCES CEREVISIAE

Genetics ◽  
1982 ◽  
Vol 102 (4) ◽  
pp. 679-690
Author(s):  
George S Zubenko ◽  
Frances J Park ◽  
Elizabeth W Jones
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Structural Gene ◽  
Enzymatic Activities ◽  
Mutant Phenotype ◽  
Yeast Cells ◽  
Carboxypeptidase Y ◽  
Nonsense Mutations

ABSTRACT Yeast cells that inherit mutations at the PEP4 locus exhibit a pronounced phenotypic lag in the expression of the mutant phenotype imparted by these mutations. This lag appears to extend to all of the enzymes that are affected by the pep4-3 mutation. For at least two of the enzymatic activities, phenotypic lag shows mitotic cosegregation. Phenotypic lag is found for meiotic progeny and for mitotic segregants from heterokaryons. The phenotypic lag in the expression of the carboxypeptidase Y deficiency is abolished by nonsense mutations in either PRC1, the structural gene for carboxypeptidase Y, or PRB1, the structural gene for proteinase B. Models to explain these observations are proposed.

scholarly journals Molecular analysis of the yeast VPS3 gene and the role of its product in vacuolar protein sorting and vacuolar segregation during the cell cycle.

1990 ◽  
Vol 111 (3) ◽  
pp. 877-892 ◽  
Author(s):  
C K Raymond ◽  
P J O'Hara ◽  
G Eichinger ◽  
J H Rothman ◽  
T H Stevens
Keyword(s):  
Selection Procedure ◽  
Temperature Shift ◽  
Yeast Cells ◽  
Carboxypeptidase Y ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Yeast Strains ◽  
Type Pattern ◽  
Mother Cells ◽  
Vacuolar Protein

vps3 mutants of the yeast Saccharomyces cerevisiae are impaired in the sorting of newly synthesized soluble vacuolar proteins and in the acidification of the vacuole (Rothman, J. H., and T. H. Stevens. Cell. 47:1041-1051; Rothman, J. H., C. T. Yamashiro, C. K. Raymond, P. M. Kane, and T. H. Stevens. 1989. J. Cell Biol. 109:93-100). The VPS3 gene, which was cloned using a novel selection procedure, encodes a low abundance, hydrophilic protein of 117 kD that most likely resides in the cytoplasm. Yeast strains bearing a deletion of the VPS3 gene (vps3-delta 1) are viable, yet their growth rate is significantly reduced relative to wild-type cells. Temperature shift experiments with strains carrying a temperature conditional vps3 allele demonstrate that cells rapidly lose the capacity to sort the vacuolar protein carboxypeptidase Y upon loss of VPS3 function. Vacuolar morphology was examined in wild-type and vps3-delta 1 yeast strains by fluorescence microscopy. The vacuoles in wild-type yeast cells are morphologically complex, and they appear to be actively partitioned between mother cells and buds during an early phase of bud growth. Vacuolar morphology in vps3-delta 1 mutants is significantly altered from the wild-type pattern, and the vacuolar segregation process seen in wild-type strains is defective in these mutants. With the exception of a vacuolar acidification defect, the phenotypes of vps3-delta 1 strains are significantly different from those of mutants lacking the vacuolar proton-translocating ATPase. These data demonstrate that the acidification defect in vps3-delta 1 cells is not the primary cause of the pleiotropic defects in vacuolar function observed in these mutants.

Combined application of monoclonal antibody to human la antigens and avidinblotin-peroxidase staining method in immunoelectron microscopy

1984 ◽  
Vol 42 ◽  
pp. 258-259
Author(s):  
K.C. Feng-Chen ◽  
B.F. Chen ◽  
A.K. Ng
Keyword(s):  
Monoclonal Antibody ◽  
Immunoelectron Microscopy ◽  
Staining Method ◽  
Ultrastructural Level ◽  
Combined Application ◽  
Antibody Method ◽  
Tissue Antigens ◽  
Pap Method ◽  
Abc Method ◽  
Unlabeled Antibody

Immunological detection of cellular and tissue antigens have been based on the widely used original enzyme labeled antibody method or the unlabeled antibody, peroxidase-anti-peroxidase (PAP) method. Recently, an improved immunoenzymatic technique for light microscopy using the avidin-biotinperoxidase complex (ABC) system was developed for cell and tissue staining, and found to be superior to the PAP technique in terms of sensitivity and specificity. The application of the ABC method in immunoelectron microscopy (IEM), however, has not been extensively evaluated. This study demonstrates that the ABC method is suitable for localizing specific cellular antigen at the ultrastructural level with monoclonal antibody (MAb), the production of which by somatic cell hybridization is now a well established procedure.

scholarly journals The plant vacuolar protein, phytohemagglutinin, is transported to the vacuole of transgenic yeast.

1987 ◽  
Vol 105 (5) ◽  
pp. 1971-1979 ◽  
Author(s):  
B W Tague ◽  
M J Chrispeels
Keyword(s):  
Signal Peptide ◽  
Yeast Cells ◽  
Cell Fractionation ◽  
Animal Cells ◽  
Yeast Vacuole ◽  
Transgenic Yeast ◽  
Protein Storage Vacuoles ◽  
Membrane Bound ◽  
Vacuolar Sorting ◽  
Vacuolar Protein

Phytohemagglutinin (PHA), the major seed lectin of the common bean, Phaseolus vulgaris, accumulates in the parenchyma cells of the cotyledons. It has been previously shown that PHA is cotranslationally inserted into the endoplasmic reticulum with cleavage of the NH2-terminal signal peptide. Two N-linked oligosaccharide side chains are added, one of which is modified to a complex type in the Golgi apparatus. PHA is then deposited in membrane-bound protein storage vacuoles which are biochemically and functionally equivalent to the vacuoles of yeast cells and the lysosomes of animal cells. We wished to determine whether yeast cells would recognize the vacuolar sorting determinant of PHA and target the protein to the yeast vacuole. We have expressed the gene for leukoagglutinating PHA (PHA-L) in yeast under control of the yeast acid phosphatase (PHO5) promoter. Under control of this promoter, PHA-L accumulates to 0.1% of the total yeast protein. PHA-L produced in yeast is glycosylated as expected for a yeast vacuolar glycoprotein. Cell fractionation studies show that PHA-L is efficiently transported to the yeast vacuole. This is the first demonstration that vacuolar targeting information is recognized between two highly divergent species. A small proportion of yeast PHA-L is secreted which may be due to inefficient recognition of the vacuolar sorting signal because of the presence of an uncleaved signal peptide on a subset of the PHA-L polypeptides. This system can now be used to identify the vacuolar sorting determinant of a plant vacuolar protein.

scholarly journals Msn2/4 regulate expression of glycolytic enzymes and control transition from quiescence to growth

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Zheng Kuang ◽  
Sudarshan Pinglay ◽  
Hongkai Ji ◽  
Jef D Boeke
Keyword(s):  
Stress Response ◽  
Glycolytic Enzymes ◽  
Yeast Cells ◽  
Quiescent State ◽  
Acetyl Coa ◽  
Response Factors ◽  
General Stress Response ◽  
Growth State ◽  
Genes Encoding ◽  
And Control

Nutrient availability and stresses impact a cell’s decision to enter a growth state or a quiescent state. Acetyl-CoA stimulates cell growth under nutrient-limiting conditions, but how cells generate acetyl-CoA under starvation stress is less understood. Here, we show that general stress response factors, Msn2 and Msn4, function as master transcriptional regulators of yeast glycolysis via directly binding and activating genes encoding glycolytic enzymes. Yeast cells lacking Msn2 and Msn4 exhibit prevalent repression of glycolytic genes and a significant delay of acetyl-CoA accumulation and reentry into growth from quiescence. Thus Msn2/4 exhibit a dual role in activating carbohydrate metabolism genes and stress response genes. These results suggest a possible mechanism by which starvation-induced stress response factors may prime quiescent cells to reenter growth through glycolysis when nutrients are limited.

Morphology of mitochondria in a teleost, salmo gairdneri

1986 ◽  
Vol 44 ◽  
pp. 194-195
Author(s):  
V. Henderson ◽  
M. J. Song
Keyword(s):  
Ultrastructural Level ◽  
High Energy ◽  
Yeast Cells ◽  
Energy Requirements ◽  
Salmo Gairdneri ◽  
Metabolic Condition ◽  
Pathological Conditions ◽  
Single Mitochondrion

Mitochondria have been observed at the ultrastructural level as spherical, oval, or sausagelike. Mitochondria average 0.3 to 1.0 um in diameter and 1.0 to 10.0 μm in length. Mitochondria may exceed these dimensions under certian physiological or pathological conditions. The number of mitochondria may reflect the metabolic condition of cells. Cells with high ATP demands display a large number of mitochondria. High energy requirements characterize muscles in both vertebrates and invertebrates. It has been established that yeast cells have but a single mitochondrion. This investigation was designed to ascertain if the numerous mitochondrial profiles represent a single mitochondrion in vertebrate cells.

Transient ER retention as stress response: conformational repair of heat-damaged proteins to secretion-competent structures

1998 ◽  
Vol 111 (11) ◽  
pp. 1575-1582
Author(s):  
N. Saris ◽  
M. Makarow
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Stress Response ◽  
Stress Condition ◽  
Cellular Stress Response ◽  
Yeast Cells ◽  
Carboxypeptidase Y ◽  
Beta Lactamase ◽  
Cell Wall Invertase ◽  
Er Retention ◽  
As Stress

Mechanisms to acquire tolerance against heat, an important environmental stress condition, have evolved in all organisms, but are largely unknown. When Saccharomyces cerevisiae cells are pre-conditioned at 37 degrees C, they survive an otherwise lethal exposure to 48–50 degrees C, and form colonies at 24 degrees C. We show here that incubation of yeast cells at 48–50 degrees C, after pre-conditioning at 37 degrees C, resulted in inactivation of exocytosis, and in conformational damage and loss of transport competence of proteins residing in the endoplasmic reticulum (ER). Soon after return of the cells to 24 degrees C, membrane traffic was resumed, but cell wall invertase, vacuolar carboxypeptidase Y and a secretory beta-lactamase fusion protein remained in the ER for different times. Thereafter their transport competence was resumed very slowly with widely varying kinetics. While the proteins were undergoing conformational repair in the ER, their native counterparts, synthesized after shift of the cells to 24 degrees C, folded normally, by-passed the heat-affected copies and exited rapidly the ER. The Hsp70 homolog Lhs1p was required for acquisition of secretion competence of heat-damaged proteins. ER retention and refolding of heat-denatured glycoproteins appear to be part of the cellular stress response.

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