Three dimensional configuration of the secretory pathway and segregation of secretion granules in the yeast Saccharomyces cerevisiae

2001 ◽  
Vol 114 (12) ◽  
pp. 2231-2239 ◽  
Author(s):  
Alain Rambourg ◽  
Catherine L. Jackson ◽  
Yves Clermont
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Golgi Apparatus ◽  
Mammalian Cells ◽  
Secretory Pathway ◽  
Secretory Granules ◽  
Staining Intensity ◽  
Permissive Temperature ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Secretion Granules

The structural elements of the secretory pathway in the budding yeast Saccharomyces cerevisiae were analyzed by 3D stereo-electron microscopy using relatively thick sections in which membranes were selectively impregnated. In a wild-type strain, tubular networks of various sizes and staining properties were distributed throughout the cytoplasm. As a rule, wide-meshed, lightly stained polygonal networks were connected to more or less fenestrated sheets of endoplasmic reticulum (ER). Some of these networks were continuous with more intensely stained networks and narrower meshes that displayed at their intersections nodular dilations that progressively increased in size and staining properties to reach those of secretion granules. Such networks presumably corresponded to Golgi elements. Indeed, stacked cisternae typical of the mammalian Golgi apparatus are rarely found in wild-type cells. However, if it is assumed that the Golgi apparatus plays a key role in the segregation and maturation of secretion granules, then tubular networks with nodular dilations should be equivalent to parts of this organelle. In correlation with the increase in size and density of the nodules there was a decrease in diameter and staining intensity of the interconnecting tubules. These results parallel observations on the formation of secretory granules in mammalian cells and suggest that the segregation of secretory material is concomitant with the progressive perforation and tubulization of previously unperforated sheets. When the sec21-3 thermosensitive mutant was examined at the nonpermissive temperature (37°C), the secretory pathway was blocked at exit from the ER, which started to accumulate as clusters of narrow, anastomosed, unperforated ribbon-like elements. When the block was released by shifting down to permissive temperature (24°C), tubular networks of various sizes and caliber, presumably Golgi in nature, formed as soon as 5 minutes after release of the block. At later time intervals, granules of various sizes and densities appeared to be released by rupture of these tubular networks or even to form at the edges of ER fenestrae. These observations support a dynamic maturation process in which the formation of secretion granules occurs by means of an oriented series of membrane transformations starting at the ER and culminating with the liberation of secretion granules from Golgi networks.

scholarly journals Smy1p, a Kinesin-related Protein That Does Not Require Microtubules

1998 ◽  
Vol 140 (4) ◽  
pp. 873-883 ◽  
Author(s):  
S.H. Lillie ◽  
S.S. Brown
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Motor Activity ◽  
Secretory Pathway ◽  
Genetic Interaction ◽  
Related Protein ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Functional Link ◽  
Specific Step ◽  
Tubulin Mutations

Abstract. We have previously reported that a defect in Myo2p, a myosin in budding yeast (Saccharomyces cerevisiae), can be partially corrected by overexpression of Smy1p, which is by sequence a kinesin-related protein (Lillie, S.H., and S.S. Brown. 1992. Nature. 356:358– 361). Such a functional link between putative actin- and microtubule-based motors is surprising, so here we have tested the prediction that Smy1p indeed acts as a microtubule-based motor. Unexpectedly, we found that abolition of microtubules by nocodazole does not interfere with the ability of Smy1p to correct the mutant Myo2p defect, nor does it interfere with the ability of Smy1p to localize properly. In addition, other perturbations of microtubules, such as treatment with benomyl or introduction of tubulin mutations, do not exacerbate the Myo2p defect. Furthermore, a mutation in SMY1 strongly predicted to destroy motor activity does not destroy Smy1p function. We have also observed a genetic interaction between SMY1 and two of the late SEC mutations, sec2 and sec4. This indicates that Smy1p can play a role even when Myo2p is wild type, and that Smy1p acts at a specific step of the late secretory pathway. We conclude that Smy1p does not act as a microtubule-based motor to localize properly or to compensate for defective Myo2p, but that it must instead act in some novel way.

scholarly journals Ascospore Formation in the Yeast Saccharomyces cerevisiae

2005 ◽  
Vol 69 (4) ◽  
pp. 565-584 ◽  
Author(s):  
Aaron M. Neiman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membranes ◽  
Secretory Pathway ◽  
Diploid Cell ◽  
Spore Wall ◽  
Second Phase ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Membrane Compartments ◽  
Two Phases

SUMMARY Sporulation of the baker's yeast Saccharomyces cerevisiae is a response to nutrient depletion that allows a single diploid cell to give rise to four stress-resistant haploid spores. The formation of these spores requires a coordinated reorganization of cellular architecture. The construction of the spores can be broadly divided into two phases. The first is the generation of new membrane compartments within the cell cytoplasm that ultimately give rise to the spore plasma membranes. Proper assembly and growth of these membranes require modification of aspects of the constitutive secretory pathway and cytoskeleton by sporulation-specific functions. In the second phase, each immature spore becomes surrounded by a multilaminar spore wall that provides resistance to environmental stresses. This review focuses on our current understanding of the cellular rearrangements and the genes required in each of these phases to give rise to a wild-type spore.

scholarly journals U1 small nuclear ribonucleoprotein particle-protein interactions are revealed in Saccharomyces cerevisiae by in vivo competition assays.

1993 ◽  
Vol 13 (4) ◽  
pp. 2126-2133 ◽  
Author(s):  
F Stutz ◽  
X C Liao ◽  
M Rosbash
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Growth Rate ◽  
Mammalian Cells ◽  
Wild Type ◽  
Ribonucleoprotein Particle ◽  
Yeast Saccharomyces Cerevisiae ◽  
Small Nuclear Ribonucleoprotein ◽  
U1 Snrna ◽  
Nuclear Ribonucleoprotein ◽  
Small Nuclear Ribonucleoprotein Particle

Two highly conserved regions of the 586-nucleotide yeast (Saccharomyces cerevisiae) U1 small nuclear RNA (snRNA) can be mutated or deleted with little or no effect on growth rate: the universally conserved loop II (corresponding to the metazoan A loop) and the yeast core region (X. Liao, L. Kretzner, B. Séraphin, and M. Rosbash, Genes Dev. 4:1766-1774, 1990). To examine the contribution of these regions to U1 small nuclear ribonucleoprotein particle (snRNP) activity, a competitor U1 gene, encoding a nonfunctional U1 snRNA molecule, was introduced into a number of strains carrying a U1 snRNA gene with loop II or yeast core mutations. The presence of the nonfunctional U1 gene lowered the growth rate of these mutant strains but not wild-type strains, consistent with the notion that mutant U1 RNAs are less active than wild-type U1 snRNAs. A detailed analysis of the U1 snRNA levels and half-lives in a number of merodiploid strains suggests that these mutant U1 snRNAs interact with U1 snRNP proteins less well than do their wild-type counterparts. Competition for protein factors during snRNP assembly could account for a number of previous observations in both yeast and mammalian cells.

scholarly journals MODULATION IN NUCLEOSIDE DIPHOSPHATASE ACTIVITY OF MAMMOTROPHIC CELLS OF THE RAT ADENOHYPOPHYSIS DURING SECRETION

1970 ◽  
Vol 18 (4) ◽  
pp. 237-250 ◽  
Author(s):  
ROBERT E. SMITH ◽  
MARILYN G. FARQUHAR
Keyword(s):  
Golgi Apparatus ◽  
Protein Secretion ◽  
Outer Surface ◽  
Secretory Granules ◽  
Hormone Secretion ◽  
Staining Intensity ◽  
Female Rat ◽  
Intercellular Spaces ◽  
Secretion Granules ◽  
Nucleoside Diphosphatase

The modulations in nucleoside diphosphatase (NDPase) activity which occur during protein secretion have been investigated in mammotrophic hormone-producing cells (MT) of the female rat adenohypophysis. Pituitaries were fixed by perfusion or immersion in glutaraldehyde, and nonfrozen sections were incubated for NDPase. Tissue was examined from lactating and estrogen-treated animals in which mammotrophic hormone secretion (MTH) is high and from postlactating rats in which secretion of MTH is suppressed. In all experimental groups, NDPase reaction product was found: ( a) in Golgi cisternae, ( b) around forming and mature secretion granules, ( c) in pericapillary and intercellular spaces and ( d) along the outer surface of endothelial and MT cell membranes. The staining intensity of both the intracellular and extracellular sites paralleled the level of MTH secretion; it was greatest after estrogen treatment, moderate during lactation and minimal in the postlactating animal. In animals given estrogen, the Golgi apparatus was increased in size and up to six successive cisternae along the concave Golgi face were filled with reaction product in virtually every cell. In postlactating animals, the Golgi apparatus was small, and there was only patchy staining of a few cisternae in some cells. From its location and fluctuations with secretion, it seems likely that NDPase activity may be associated with the condensation and/or discharge of secretory granules.

U1 small nuclear ribonucleoprotein particle-protein interactions are revealed in Saccharomyces cerevisiae by in vivo competition assays

1993 ◽  
Vol 13 (4) ◽  
pp. 2126-2133
Author(s):  
F Stutz ◽  
X C Liao ◽  
M Rosbash
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Growth Rate ◽  
Mammalian Cells ◽  
Wild Type ◽  
Ribonucleoprotein Particle ◽  
Yeast Saccharomyces Cerevisiae ◽  
Small Nuclear Ribonucleoprotein ◽  
U1 Snrna ◽  
Nuclear Ribonucleoprotein ◽  
Small Nuclear Ribonucleoprotein Particle

Two highly conserved regions of the 586-nucleotide yeast (Saccharomyces cerevisiae) U1 small nuclear RNA (snRNA) can be mutated or deleted with little or no effect on growth rate: the universally conserved loop II (corresponding to the metazoan A loop) and the yeast core region (X. Liao, L. Kretzner, B. Séraphin, and M. Rosbash, Genes Dev. 4:1766-1774, 1990). To examine the contribution of these regions to U1 small nuclear ribonucleoprotein particle (snRNP) activity, a competitor U1 gene, encoding a nonfunctional U1 snRNA molecule, was introduced into a number of strains carrying a U1 snRNA gene with loop II or yeast core mutations. The presence of the nonfunctional U1 gene lowered the growth rate of these mutant strains but not wild-type strains, consistent with the notion that mutant U1 RNAs are less active than wild-type U1 snRNAs. A detailed analysis of the U1 snRNA levels and half-lives in a number of merodiploid strains suggests that these mutant U1 snRNAs interact with U1 snRNP proteins less well than do their wild-type counterparts. Competition for protein factors during snRNP assembly could account for a number of previous observations in both yeast and mammalian cells.

scholarly journals Replacement of Lys by Glu in a transmembrane segment strongly impairs the function of the uracil permease from Saccharomyces cerevisiae

1995 ◽  
Vol 308 (3) ◽  
pp. 847-851 ◽  
Author(s):  
D Urban-Grimal ◽  
B Pinson ◽  
J Chevallier ◽  
R Haguenauer-Tsapis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Secretory Pathway ◽  
Fold Increase ◽  
Recent Experimental Data ◽  
Spontaneous Mutant ◽  
Wild Type ◽  
Transmembrane Segment ◽  
Yeast Saccharomyces Cerevisiae ◽  
Equilibrium Binding

The co-transport of uracil and protons through the plasma membrane of the yeast Saccharomyces cerevisiae is mediated by a specific permease encoded by the FUR4 gene. The uracil permease is a multi-spanning membrane protein that follows the secretory pathway to the plasma membrane. Recent experimental data led to the proposal of a two-dimensional model of its topology. A spontaneous mutant corresponding to the substitution of Lys-272 by glutamic acid was obtained. The influence of this mutation was studied by comparing the wild-type and mutant permeases produced in a strain carrying a chromosomal deletion of the FUR4 gene. The mutant permease is correctly targeted to the plasma membrane and its stability is similar to that of the wild-type permease. The uptake parameters for the mutant permease were impaired and showed an approximately 65-fold increase of apparent K(m) and a decrease in apparent Vmax. Equilibrium binding measurements with enriched plasma membrane preparations showed an approximately 70-fold increase in apparent Kd in the mutant, whereas its Bmax. was similar to that of the wild type. Lys-272 is fully conserved in the uracil permease family and is predicted to lie in the fourth transmembrane segment of the protein. It seems to be essential for both efficient uracil binding and translocation.

scholarly journals The functioning of mammalian ClC-2 chloride channel in Saccharomyces cerevisiae cells requires an increased level of Kha1p

2005 ◽  
Vol 390 (3) ◽  
pp. 655-664 ◽  
Author(s):  
Krzysztof Flis ◽  
Alexandre Hinzpeter ◽  
Aleksander Edelman ◽  
Anna Kurlandzka
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Golgi Apparatus ◽  
Chloride Channel ◽  
Mammalian Cells ◽  
Chloride Channels ◽  
Functional Expression ◽  
Ph Control ◽  
Gene Encoding ◽  
General Applicability ◽  
Yeast Saccharomyces Cerevisiae

The mammalian chloride channel ClC-2 is a member of the CLC voltage-gated chloride channels family. This broadly expressed protein shows diverse cellular locations and despite numerous studies, its precise function is poorly understood. Disruption of ClC-2-encoding gene in mouse leads to retinal and testicular degeneration and mutations in CLC2 (gene encoding the ClC-2 channel) are associated with idiopathic generalized epilepsies. ClC-2 may also be responsible for Cl− transport in mouse salivary glands. The only CLC homologue of the yeast Saccharomyces cerevisiae, Gef1p, exhibits CLC activity. We expressed the mammalian ClC-2 protein in S. cerevisiae devoid of Gef1p in an attempt to identify yeast proteins influencing the functioning of ClC-2. The presence of such proteins in yeast could indicate the existence of their homologues in mammalian cells and would greatly aid their identification. Expression of ClC-2 in yeast required optimization of the sequence context of the AUG translation initiation codon. After obtaining an efficient translation, we found that rat ClC-2 cannot directly substitute for yeast Gef1p. Functional substitution for Gef1p was, however, achieved in the presence of an increased level of intact or C-terminally truncated yeast Kha1 protein. Based on the deduced amino acid sequence, the Kha1 protein can be classified as a Na+/H+ transporter since it has a large N-terminal domain similar to the family of NHEs (Na+/H+ exchangers). This suggests that the Kha1p may take part in the regulation of intracellular cation homoeostasis and pH control. We have established that Kha1p is localized in the same cellular compartment as Gef1p and yeast-expressed ClC-2: the Golgi apparatus. We propose that Kha1p may aid ClC-2-dependent suppression of the Δgef1-assocciated growth defects by keeping the Golgi apparatus pH in a range suitable for ClC-2 activity. The approach employed in the present study may be of general applicability to the characterization of poorly understood proteins by their functional expression in yeast.

scholarly journals Nonrecombinant meiosis I nondisjunction in Saccharomyces cerevisiae induced by tRNA ochre suppressors.

Genetics ◽  
1989 ◽  
Vol 123 (1) ◽  
pp. 81-95 ◽  
Author(s):  
E J Louis ◽  
J E Haber
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mitotic Chromosome ◽  
Stop Codon ◽  
Fold Increase ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Nonsense Mutations ◽  
Chromosome Iii ◽  
Meiosis I ◽  
Chromosome Nondisjunction

Abstract The presence of the tRNA ochre suppressors SUP11 and SUP5 is found to induce meiosis I nondisjunction in the yeast Saccharomyces cerevisiae. The induction increases with increasing dosage of the suppressor and decreases in the presence of an antisuppressor. The effect is independent of the chromosomal location of SUP11. Each of five different chromosomes monitored exhibited nondisjunction at frequencies of 0.1%-1.1% of random spores, which is a 16-160-fold increase over wild-type levels. Increased nondisjunction is reflected by a marked increase in tetrads with two and zero viable spores. In the case of chromosome III, for which a 50-cM map interval was monitored, the resulting disomes are all in the parental nonrecombinant configuration. Recombination along chromosome III appears normal both in meioses that have no nondisjunction and in meioses for which there was nondisjunction of another chromosome. We propose that a proportion of one or more proteins involved in chromosome pairing, recombination or segregation are aberrant due to translational read-through of the normal ochre stop codon. Hygromycin B, an antibiotic that can suppress nonsense mutations via translational read-through, also induces nonrecombinant meiosis I nondisjunction. Increases in mistranslation, therefore, increase the production of aneuploids during meiosis. There was no observable effect of SUP11 on mitotic chromosome nondisjunction; however some disomes caused SUP11 ade2-ochre strains to appear white or red, instead of pink.

scholarly journals Rsp5p, a New Link between the Actin Cytoskeleton and Endocytosis in the Yeast Saccharomyces cerevisiae

2002 ◽  
Vol 22 (20) ◽  
pp. 6946-6948 ◽  
Author(s):  
Joanna Kamińska ◽  
Beata Gajewska ◽  
Anita K. Hopper ◽  
Teresa ˙Zołądek
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Actin Cytoskeleton ◽  
Cytoskeletal Proteins ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Ww Domains ◽  
Ubiquitin Protein Ligase ◽  
Growth Defects ◽  
Genes Encoding ◽  
Cytoskeleton Dynamics

ABSTRACT Rsp5p is an ubiquitin-protein ligase of Saccharomyces cerevisiae that has been implicated in numerous processes including transcription, mitochondrial inheritance, and endocytosis. Rsp5p functions at multiple steps of endocytosis, including ubiquitination of substrates and other undefined steps. We propose that one of the roles of Rsp5p in endocytosis involves maintenance and remodeling of the actin cytoskeleton. We report the following. (i) There are genetic interactions between rsp5 and several mutant genes encoding actin cytoskeletal proteins. rsp5 arp2, rsp5 end3, and rsp5 sla2 double mutants all show synthetic growth defects. Overexpressed wild-type RSP5 or mutant rsp5 genes with lesions of some WW domains suppress growth defects of arp2 and end3 cells. The defects in endocytosis, actin cytoskeleton, and morphology of arp2 are also suppressed. (ii) Rsp5p and Sla2p colocalize in abnormal F-actin-containing clumps in arp2 and pan1 mutants. Immunoprecipitation experiments confirmed that Rsp5p and Act1p colocalize in pan1 mutants. (iii) Rsp5p and Sla2p coimmunoprecipitate and partially colocalize to punctate structures in wild-type cells. These studies provide the first evidence for an interaction of an actin cytoskeleton protein with Rsp5p. (iv) rsp5-w1 mutants are resistant to latrunculin A, a drug that sequesters actin monomers and depolymerizes actin filaments, consistent with the fact that Rsp5p is involved in actin cytoskeleton dynamics.

scholarly journals The Highly Conserved tRNAHis Guanylyltransferase Thg1p Interacts with the Origin Recognition Complex and Is Required for the G2/M Phase Transition in the Yeast Saccharomyces cerevisiae

2005 ◽  
Vol 4 (4) ◽  
pp. 832-835 ◽  
Author(s):  
Terri S. Rice ◽  
Min Ding ◽  
David S. Pederson ◽  
Nicholas H. Heintz
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Origin Recognition Complex ◽  
Nuclear Division ◽  
Permissive Temperature ◽  
Yeast Saccharomyces Cerevisiae ◽  
M Phase ◽  
And Migration ◽  
Origin Recognition

ABSTRACT Here we show that the Saccharomyces cerevisiae tRNAHis guanylyltransferase Thg1p interacts with the origin recognition complex in vivo and in vitro and that overexpression of hemagglutinin-Thg1p selectively impedes growth of orc2-1(Ts) cells at the permissive temperature. Studies with conditional mutants indicate that Thg1p couples nuclear division and migration to cell budding and cytokinesis in yeast.

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