scholarly journals Yeast KRE2 defines a new gene family encoding probable secretory proteins, and is required for the correct N-glycosylation of proteins.

Genetics ◽  
1992 ◽  
Vol 130 (2) ◽  
pp. 273-283 ◽  
Author(s):  
K Hill ◽  
C Boone ◽  
M Goebl ◽  
R Puccia ◽  
A M Sdicu ◽  
...  
Keyword(s):  
Gene Family ◽  
Secretory Pathway ◽  
Signal Sequence ◽  
Secretory Proteins ◽  
Chromosome 15 ◽  
Amino Acid Residues ◽  
New Gene ◽  
New Yeast ◽  
Average Size

Abstract We have cloned, sequenced and disrupted the KRE2 gene of Saccharomyces cerevisiae, identified by killer-resistant mutants with a defective cell wall receptor for the toxin. The KRE2 gene is close to PHO8 on chromosome 4, and encodes a predicted 49-kD protein, Kre2p, that probably enters the secretory pathway. Haploid cells carrying a disruption of the KRE2 locus grow more slowly than wild-type cells at 30 degrees, and fail to grow at 37 degrees. At 30 degrees, kre2 mutants showed altered N-linked glycosylation of proteins, as the average size of N-linked outer chains was reduced. We identified two other genes, YUR1 on chromosome 10, and KTR1 on chromosome 15, whose predicted products share 36% identity with Kre2p over more than 300 amino acid residues. Yur1p has an N-terminal signal sequence like Kre2p, while Ktr1p has a predicted topology consistent with a type 2 membrane protein. In all cases the conserved regions of these proteins appear to be on the lumenal side of secretory compartments, suggesting related function. KRE2, KTR1 and YUR1 define a new yeast gene family.

scholarly journals Analysis of Protein Localization and Secretory Pathway Function Using the Yeast Saccharomyces cerevisiae

2002 ◽  
Vol 1 (4) ◽  
pp. 173-192 ◽  
Author(s):  
Elizabeth Vallen
Keyword(s):  
Cell Biology ◽  
Secretory Pathway ◽  
Signal Sequence ◽  
Protein Localization ◽  
Chimeric Protein ◽  
Secretory Proteins ◽  
Yeast Saccharomyces Cerevisiae ◽  
Isolation And Characterization ◽  
Yeast Transformants ◽  
Histidinol Dehydrogenase

The isolation and characterization of mutants has been crucial in understanding a number of processes in the field of cell biology. In this exercise, students examine the effects of mutations in the secretory pathway on protein localization. Yeast strains deficient for synthesis of histidinol dehydrogenase are transformed with a plasmid encoding a chimeric protein. The chimera contains a signal sequence fused to histidinol dehydrogenase. A strain with a defect in the translocation of secretory proteins into the endoplasmic reticulum (ER) accumulates sufficient histidinol dehydrogenase in the cytoplasm to grow on media lacking histidine. In contrast, yeast proficient for secretion, or yeast with secretion defects later in the pathway, are unable to grow on media lacking histidine. Student analysis of the experimental yeast transformants and appropriate controls allows investigation into the effects of conditional defects in the secretory pathway on both cell viability and protein localization. The exercise is usually performed in a manner that allows students to execute a number of techniques common in molecular biology laboratories, including plasmid minipreps, restriction digestions, and Southern blots. Student understanding and enjoyment of the exercise was assessed by laboratory reports, oral and written examinations, and questionnaires. After completion of these experiments, students can describe the utility of protein fusions, the roles of mutant analysis in cell biology, and the steps taken by proteins transiting the secretory pathway.

scholarly journals Early stages of functional diversification in the Rab GTPase gene family revealed by genomic and localization studies in Paramecium species

2017 ◽  
Vol 28 (8) ◽  
pp. 1101-1110 ◽  
Author(s):  
Lydia J. Bright ◽  
Jean-Francois Gout ◽  
Michael Lynch
Keyword(s):  
Gene Family ◽  
Gene Families ◽  
Rab Gtpase ◽  
Rab Proteins ◽  
Whole Genome ◽  
Amino Acid Residues ◽  
Functional Diversification ◽  
Whole Genome Duplications ◽  
Genome Duplications ◽  
New Gene

New gene functions arise within existing gene families as a result of gene duplication and subsequent diversification. To gain insight into the steps that led to the functional diversification of paralogues, we tracked duplicate retention patterns, expression-level divergence, and subcellular markers of functional diversification in the Rab GTPase gene family in three Paramecium aurelia species. After whole-genome duplication, Rab GTPase duplicates are more highly retained than other genes in the genome but appear to be diverging more rapidly in expression levels, consistent with early steps in functional diversification. However, by localizing specific Rab proteins in Paramecium cells, we found that paralogues from the two most recent whole-genome duplications had virtually identical localization patterns, and that less closely related paralogues showed evidence of both conservation and diversification. The functionally conserved paralogues appear to target to compartments associated with both endocytic and phagocytic recycling functions, confirming evolutionary and functional links between the two pathways in a divergent eukaryotic lineage. Because the functionally diversifying paralogues are still closely related to and derived from a clade of functionally conserved Rab11 genes, we were able to pinpoint three specific amino acid residues that may be driving the change in the localization and thus the function in these proteins.

scholarly journals The PBN1 Gene of Saccharomyces cerevisiae: An Essential Gene That Is Required for the Post-translational Processing of the Protease B Precursor

Genetics ◽  
1998 ◽  
Vol 149 (3) ◽  
pp. 1277-1292 ◽  
Author(s):  
Rajesh R Naik ◽  
Elizabeth W Jones
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Secretory Pathway ◽  
Transmembrane Domain ◽  
Essential Gene ◽  
Signal Sequence ◽  
Signal Peptidase ◽  
Amino Terminal ◽  
Autocatalytic Cleavage ◽  
Protease B ◽  
Chromosome Iii

Abstract The vacuolar hydrolase protease B in Saccharomyces cerevisiae is synthesized as an inactive precursor (Prb1p). The precursor undergoes post-translational modifications while transiting the secretory pathway. In addition to N- and O -linked glycosylations, four proteolytic cleavages occur during the maturation of Prb1p. Removal of the signal peptide by signal peptidase and the autocatalytic cleavage of the large aminoterminal propeptide occur in the endoplasmic reticulum (ER). Two carboxy-terminal cleavages of the post regions occur in the vacuole: the first cleavage is catalyzed by protease A and the second results from autocatalysis. We have isolated a mutant, pbn1-1, that exhibits a defect in the ER processing of Prb1p. The autocatalytic cleavage of the propeptide from Prb1p does not occur and Prb1p is rapidly degraded in the cytosol. PBN1 was cloned and is identical to YCL052c on chromosome III. PBN1 is an essential gene that encodes a novel protein. Pbn1p is predicted to contain a sub-C-terminal transmembrane domain but no signal sequence. A functional HA epitope-tagged Pbn1p fusion localizes to the ER. Pbn1p is N-glycosylated in its amino-terminal domain, indicating a lumenal orientation despite the lack of a signal sequence. Based on these results, we propose that one of the functions of Pbn1p is to aid in the autocatalytic processing of Prb1p.

scholarly journals The unfolded protein response coordinates the production of endoplasmic reticulum protein and endoplasmic reticulum membrane.

1997 ◽  
Vol 8 (9) ◽  
pp. 1805-1814 ◽  
Author(s):  
J S Cox ◽  
R E Chapman ◽  
P Walter
Keyword(s):  
Endoplasmic Reticulum ◽  
Unfolded Protein Response ◽  
Secretory Pathway ◽  
Lipid Synthesis ◽  
Secretory Proteins ◽  
Endoplasmic Reticulum Membrane ◽  
Yeast Saccharomyces Cerevisiae ◽  
Unfolded Protein ◽  
The Unfolded Protein Response ◽  
Protein Response

The endoplasmic reticulum (ER) is a multifunctional organelle responsible for production of both lumenal and membrane components of secretory pathway compartments. Secretory proteins are folded, processed, and sorted in the ER lumen and lipid synthesis occurs on the ER membrane itself. In the yeast Saccharomyces cerevisiae, synthesis of ER components is highly regulated: the ER-resident proteins by the unfolded protein response and membrane lipid synthesis by the inositol response. We demonstrate that these two responses are intimately linked, forming different branches of the same pathway. Furthermore, we present evidence indicating that this coordinate regulation plays a role in ER biogenesis.

scholarly journals Cofilin recruits F-actin to SPCA1 and promotes Ca2+-mediated secretory cargo sorting

2014 ◽  
Vol 206 (5) ◽  
pp. 635-654 ◽  
Author(s):  
Christine Kienzle ◽  
Nirakar Basnet ◽  
Alvaro H. Crevenna ◽  
Gisela Beck ◽  
Bianca Habermann ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Nitrilotriacetic Acid ◽  
Calcium Atpase ◽  
Secretory Proteins ◽  
Dependent Manner ◽  
Agarose Beads ◽  
P Type ◽  
Golgi Network ◽  
Phosphorylation Domain ◽  
Cargo Sorting

The actin filament severing protein cofilin-1 (CFL-1) is required for actin and P-type ATPase secretory pathway calcium ATPase (SPCA)-dependent sorting of secretory proteins at the trans-Golgi network (TGN). How these proteins interact and activate the pump to facilitate cargo sorting, however, is not known. We used purified proteins to assess interaction of the cytoplasmic domains of SPCA1 with actin and CFL-1. A 132–amino acid portion of the SPCA1 phosphorylation domain (P-domain) interacted with actin in a CFL-1–dependent manner. This domain, coupled to nickel nitrilotriacetic acid (Ni-NTA) agarose beads, specifically recruited F-actin in the presence of CFL-1 and, when expressed in HeLa cells, inhibited Ca2+ entry into the TGN and secretory cargo sorting. Mutagenesis of four amino acids in SPCA1 that represent the CFL-1 binding site also affected Ca2+ import into the TGN and secretory cargo sorting. Altogether, our findings reveal the mechanism of CFL-1–dependent recruitment of actin to SPCA1 and the significance of this interaction for Ca2+ influx and secretory cargo sorting.

scholarly journals Membrane traffic between secretory compartments is differentially affected during mitosis.

1990 ◽  
Vol 1 (5) ◽  
pp. 415-424 ◽  
Author(s):  
T Kreiner ◽  
H P Moore
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Secretory Pathway ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Membrane Traffic ◽  
Human Growth ◽  
Secretory Proteins ◽  
Viral Membrane ◽  
Mitotic Cells

Membrane traffic has been shown to be regulated during cell division. In particular, with the use of viral membrane proteins as markers, endoplasmic reticulum (ER)-to-Golgi transport in mitotic cells has been shown to be essentially blocked. However, the effect of mitosis on other steps in the secretory pathway is less clear, because an early block makes examination of following steps difficult. Here, we report studies on the functional characteristics of secretory pathways in mitotic mammalian tissue culture cells by the use of a variety of markers. Chinese hamster ovary cells were transfected with cDNAs encoding secretory proteins. Consistent with earlier results following viral membrane proteins, we found that the overall secretory pathway is nonfunctional in mitotic cells, and a major block to secretion is at the step between ER and Golgi: the overall rate of secretion of human growth hormone is reduced at least 10-fold in mitotic cells, and export of truncated vesicular stomatitis virus G protein from the ER is inhibited to about the same extent, as judged by acquisition of endoglycosidase H resistance. To ascertain the integrity of transport from the trans-Golgi to plasma membrane, we followed the secretion of sulfated glycosaminoglycan (GAG) chains, which are synthesized in the Golgi and thus are not subject to the earlier ER-to-Golgi block. GAG chains are valid markers for the pathway taken by constitutive secretory proteins; both protein secretion and GAG chain secretion are sensitive to treatment with n-ethyl-maleimide and monensin and are blocked at 19 degrees C. We found that the extent of GAG-chain secretion is not altered during mitosis, although the initial rate of secretion is reduced about twofold in mitotic compared with interphase cells. Thus, during mitosis, transport from the trans-Golgi to plasma membrane is much less hindered than ER-to-Golgi traffic. We conclude that transport steps are not affected to the same extent during mitosis.

An ubiquitously expressed gene 3.5 kilobases upstream of the glycerol-3-phosphate dehydrogenase gene in mice

1989 ◽  
Vol 9 (3) ◽  
pp. 935-945
Author(s):  
L A Johnston ◽  
M A Kotarski ◽  
D J Jerry ◽  
L P Kozak
Keyword(s):  
Sequence Similarity ◽  
Single Copy ◽  
Transcriptional Unit ◽  
Chromosome 15 ◽  
Glycerol Phosphate ◽  
Reading Frame ◽  
Dehydrogenase Gene ◽  
New Gene ◽  
Or Gene ◽  
Glycerol 3 Phosphate Dehydrogenase

While studying the organization of the mouse glycerol-phosphate dehydrogenase gene (Gdc-1 on chromosome 15), we identified a novel transcriptional unit located only 3.4 kilobases (kb) upstream of the 5' end of the Gdc-1 gene. This gene has been provisionally named D15Kz1. The unusual proximity of these two genes led us to investigate the pattern of expression and sequence characteristics of the new gene for comparison with those of Gdc-1. D15Kz1 was found to have transcripts of 3.2 and 3.4 kb in length. The 3.4-kb transcript was expressed at low levels in all tissues examined, whereas the 3.2-kb transcript was detected only in the cerebral cortex and the brown fat. D15Kz1 and Gdc-1 are not coordinately regulated, as evidenced by the characteristics of their expression in several tissues and in differentiating 3T3-F442A adipocyte cultures. A cDNA sequence of 3,105 bases isolated from an embryonal carcinoma lambda gt10 cDNA library had a large open reading frame of 461 amino acids at one end followed by 1.6 kb of sequence with multiple stop codons. Algorithms used to search the protein and nucleic acid data bases detected no significant sequence similarity to any other protein or gene. Southern blot analysis of genomic DNA using the D15Kz1 cDNA as a probe indicated that D15Kz1 is a single-copy gene in the mouse genome and that it is conserved in humans, rats, and chickens. This conservation of gene sequences suggests that D15Kz1 encodes a protein with an important cellular function.

Secretion-defective mutations in the signal sequence for Saccharomyces cerevisiae invertase

1986 ◽  
Vol 6 (7) ◽  
pp. 2382-2391
Author(s):  
C A Kaiser ◽  
D Botstein
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Secretory Pathway ◽  
Signal Sequence ◽  
Mutant Gene ◽  
Yeast Cells ◽  
Molecular Weight Characteristic ◽  
Cell Extracts ◽  
Substitution Mutations ◽  
Membrane Components

Nine mutations in the signal sequence region of the gene specifying the secreted Saccharomyces cerevisiae enzyme invertase were constructed in vitro. The consequences of these mutations were studied after returning the mutated genes to yeast cells. Short deletions and two extensive substitution mutations allowed normal expression and secretion of invertase. Other substitution mutations and longer deletions blocked the formation of extracellular invertase. Yeast cells carrying this second class of mutant gene expressed novel active internal forms of invertase that exhibited the following properties. The new internal proteins had the mobilities in denaturing gels expected of invertase polypeptides that had retained a defective signal sequence and were otherwise unmodified. The large increase in molecular weight characteristic of glycosylation was not seen. On nondenaturing gels the mutant enzymes were found as heterodimers with a normal form of invertase that is known to be cytoplasmic, showing that the mutant forms of the enzyme are assembled in the same compartment as the cytoplasmic enzyme. All of the mutant enzymes were soluble and not associated with the membrane components after fractionation of crude cell extracts on sucrose gradients. Therefore, these signal sequence mutations result in the production of active internal invertase that has lost the ability to enter the secretory pathway. This demonstrates that the signal sequence is required for the earliest steps in membrane translocation.

Regulated and constitutive secretion of distinct molecular forms of acetylcholinesterase from PC12 cells

1993 ◽  
Vol 106 (3) ◽  
pp. 731-740 ◽  
Author(s):  
E.S. Schweitzer
Keyword(s):  
Pc12 Cells ◽  
Secretory Pathway ◽  
Intracellular Localization ◽  
Synaptic Function ◽  
Molecular Forms ◽  
Secretory Proteins ◽  
Secretory Pathways ◽  
Regulated Secretion ◽  
Constitutive Secretion ◽  
A Cell

PC12 cells secrete the enzyme acetylcholinesterase (AChE) while at rest, and increase the overall rate of this secretion 2-fold upon depolarization. This behavior is different from the release of other markers by the constitutive or regulated secretory pathways in PC12 cells. Both the resting and stimulated release of AChE are unchanged after treatment with a membrane-impermeable esterase inhibitor, demonstrating that it represents true secretion and not shedding from the cell surface. The stimulation release of AChE is Ca(2+)-dependent, while the unstimulated release is not. Analysis of the molecular forms of AChE secreted by PC12 cells indicates that the release of AChE actually involves two concurrent but independent secretory processes, and that the G4 form of the enzyme is secreted constitutively, while both the G2 and G4 forms are secreted in a regulated manner, presumably from regulated secretory vesicles. Compared with other regulated secretory proteins, a much smaller fraction of cellular AChE is secreted, and the intracellular localization of this enzyme differs from that of other regulated secretory proteins. The demonstration that a cell line that exhibits regulated secretion of acetylcholine (ACh) is also capable of regulated secretion of AChE provides additional evidence for the existence of multiple regulated secretory pathways within a single cell. Moreover, there appears to be a selective packaging of different molecular forms of AChE into the regulated versus the constitutive secretory pathway. Both the specificity of sorting of AChE and the regulation of its secretion suggest that AChE may play a more dynamic role in synaptic function than has been recognized previously.

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