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 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 The Road Less Traveled? Unconventional Protein Secretion at Parasite–Host Interfaces

2021 ◽  
Vol 9 ◽  
Author(s):  
Erina A. Balmer ◽  
Carmen Faso
Keyword(s):  
Protein Secretion ◽  
Cell Biology ◽  
Mammalian Cells ◽  
Cell Model ◽  
Signal Sequence ◽  
Model Organism ◽  
Secreted Proteins ◽  
Current Status ◽  
Secretory Proteins ◽  
Secretion Pathways

Protein secretion in eukaryotic cells is a well-studied process, which has been known for decades and is dealt with by any standard cell biology textbook. However, over the past 20 years, several studies led to the realization that protein secretion as a process might not be as uniform among different cargos as once thought. While in classic canonical secretion proteins carry a signal sequence, the secretory or surface proteome of several organisms demonstrated a lack of such signals in several secreted proteins. Other proteins were found to indeed carry a leader sequence, but simply circumvent the Golgi apparatus, which in canonical secretion is generally responsible for the modification and sorting of secretory proteins after their passage through the endoplasmic reticulum (ER). These alternative mechanisms of protein translocation to, or across, the plasma membrane were collectively termed “unconventional protein secretion” (UPS). To date, many research groups have studied UPS in their respective model organism of choice, with surprising reports on the proportion of unconventionally secreted proteins and their crucial roles for the cell and survival of the organism. Involved in processes such as immune responses and cell proliferation, and including far more different cargo proteins in different organisms than anyone had expected, unconventional secretion does not seem so unconventional after all. Alongside mammalian cells, much work on this topic has been done on protist parasites, including genera Leishmania, Trypanosoma, Plasmodium, Trichomonas, Giardia, and Entamoeba. Studies on protein secretion have mainly focused on parasite-derived virulence factors as a main source of pathogenicity for hosts. Given their need to secrete a variety of substrates, which may not be compatible with canonical secretion pathways, the study of mechanisms for alternative secretion pathways is particularly interesting in protist parasites. In this review, we provide an overview on the current status of knowledge on UPS in parasitic protists preceded by a brief overview of UPS in the mammalian cell model with a focus on IL-1β and FGF-2 as paradigmatic UPS substrates.

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 Genome scale modeling of the protein secretory pathway reveals novel targets for improved recombinant protein production in yeast

2021 ◽  
Author(s):  
Feiran Li ◽  
Yu Chen ◽  
Qi Qi ◽  
Yanyan Wang ◽  
Le Yuan ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Recombinant Proteins ◽  
Recombinant Protein Production ◽  
Protein Production ◽  
Secretory Pathway ◽  
Protein Translation ◽  
Cellular Growth ◽  
Secretory Proteins ◽  
Yeast Saccharomyces Cerevisiae ◽  
Genome Scale

Eukaryal cells are used for the production of many recombinant pharmaceutical proteins, including several of the current top-selling products. The protein secretory pathway in eukaryal cells is complex and involves many different processes such as post-translational modifications, translocation, and folding. Furthermore, recombinant protein production competes with native secretory proteins for the limited energy and proteome resources allocated to the protein secretory pathway. Due to the complexity of this pathway, improvement through metabolic engineering has traditionally been relatively ad-hoc; and considering the industrial importance of this pathway, there is a need for more systematic approaches for novel design principles. Here, we present the first proteome-constrained genome-scale protein secretory model of a eukaryal cell, namely for the yeast Saccharomyces cerevisiae (pcSecYeast). The model contains all key processes of this pathway, i.e., protein translation, modification, and degradation coupled with metabolism. The model can capture delicate phenotypic changes such as the switch in the use of specific glucose transporters in response to changing extracellular glucose concentration. Furthermore, the model can also simulate the effects of protein misfolding on cellular growth, suggesting that retro-translocation of misfolded proteins contributes to protein retention in the Endoplasmic reticulum (ER). We used pcSecYeast to simulate various recombinant proteins production and identified overexpression targets for different recombinant proteins overproduction. We experimentally validated many of the predicted targets for α-amylase production in this study, and the results show that the secretory pathways have more limited capacity than metabolism in terms of protein secretion.

scholarly journals Posttranslational processing of the prohormone-cleaving Kex2 protease in the Saccharomyces cerevisiae secretory pathway.

1991 ◽  
Vol 115 (2) ◽  
pp. 297-307 ◽  
Author(s):  
C A Wilcox ◽  
R S Fuller
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Golgi Complex ◽  
Secretory Pathway ◽  
Apparent Molecular Weight ◽  
Secretory Proteins ◽  
Core Oligosaccharide ◽  
Yeast Saccharomyces Cerevisiae ◽  
Peptide Cleavage ◽  
Amino Terminal ◽  
Kex2 Protease

The Kex2 protease of the yeast Saccharomyces cerevisiae is a prototypical eukaryotic prohormone-processing enzyme that cleaves precursors of secreted peptides at pairs of basic residues. Here we have established the pathway of posttranslational modification of Kex2 protein using immunoprecipitation of the biosynthetically pulse-labeled protein from a variety of wild-type and mutant yeast strains as the principal methodology. Kex2 protein is initially synthesized as a prepro-enzyme that undergoes cotranslational signal peptide cleavage and addition of Asn-linked core oligosaccharide and Ser/Thr-linked mannose in the ER. The earliest detectable species, I1 (approximately 129 kD), undergoes rapid amino-terminal proteolytic removal of a approximately 9-kD pro-segment yielding species I2 (approximately 120 kD) before arrival at the Golgi complex. Transport to the Golgi complex is marked by extensive elaboration of Ser/Thr-linked chains and minor modification of Asn-linked oligosaccharide. During the latter phase of its lifetime, Kex2 protein undergoes a gradual increase in apparent molecular weight. This final modification serves as a marker for association of Kex2 protease with a late compartment of the yeast Golgi complex in which it is concentrated about 27-fold relative to other secretory proteins.

scholarly journals Isolation and characterization of yeast mutants in the cytoplasm to vacuole protein targeting pathway.

1995 ◽  
Vol 131 (3) ◽  
pp. 591-602 ◽  
Author(s):  
T M Harding ◽  
K A Morano ◽  
S V Scott ◽  
D J Klionsky
Keyword(s):  
Secretory Pathway ◽  
Protein Targeting ◽  
Protein Localization ◽  
Amino Terminal ◽  
Isolation And Characterization ◽  
Alternate Mechanism ◽  
Complementation Groups ◽  
Vacuolar Protein ◽  
Yeast Mutants

In Saccharomyces cerevisiae the vacuolar protein aminopeptidase I (API) is localized to the vacuole independent of the secretory pathway. The alternate targeting mechanism used by this protein has not been characterized. API is synthesized as a 61-kD soluble cytosolic precursor. Upon delivery to the vacuole, the amino-terminal propeptide is removed by proteinase B (PrB) to yield the mature 50-kD hydrolase. We exploited this delivery-dependent maturation event in a mutant screen to identify genes whose products are involved in API targeting. Using antiserum to the API propeptide, we isolated mutants that accumulate precursor API. These mutants, designated cvt, fall into eight complementation groups, five of which define novel genes. These five complementation groups exhibit a specific defect in maturation of API, but do not have a significant effect on vacuolar protein targeting through the secretory pathway. Localization studies show that precursor API accumulates outside of the vacuole in all five groups, indicating that they are blocked in API targeting and/or translocation. Future analysis of these gene products will provide information about the subcellular components involved in this alternate mechanism of vacuolar protein localization.

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 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 SEN1, a positive effector of tRNA-splicing endonuclease in Saccharomyces cerevisiae.

1992 ◽  
Vol 12 (5) ◽  
pp. 2154-2164 ◽  
Author(s):  
D J DeMarini ◽  
M Winey ◽  
D Ursic ◽  
F Webb ◽  
M R Culbertson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Amino Acid ◽  
Gene Product ◽  
Signal Sequence ◽  
Null Alleles ◽  
Nucleoside Triphosphate ◽  
Temperature Sensitive ◽  
Yeast Saccharomyces Cerevisiae ◽  
Amino Terminal

The SEN1 gene, which is essential for growth in the yeast Saccharomyces cerevisiae, is required for endonucleolytic cleavage of introns from all 10 families of precursor tRNAs. A mutation in SEN1 conferring temperature-sensitive lethality also causes in vivo accumulation of pre-tRNAs and a deficiency of in vitro endonuclease activity. Biochemical evidence suggests that the gene product may be one of several components of a nuclear-localized splicing complex. We have cloned the SEN1 gene and characterized the SEN1 mRNA, the SEN1 gene product, the temperature-sensitive sen1-1 mutation, and three SEN1 null alleles. The SEN1 gene corresponds to a 6,336-bp open reading frame coding for a 2,112-amino-acid protein (molecular mass, 239 kDa). Using antisera directed against the C-terminal end of SEN1, we detect a protein corresponding to the predicted molecular weight of SEN1. The SEN1 protein contains a leucine zipper motif, consensus elements for nucleoside triphosphate binding, and a potential nuclear localization signal sequence. The carboxy-terminal 1,214 amino acids of the SEN1 protein are essential for growth, whereas the amino-terminal 898 amino acids are dispensable. A sequence of approximately 500 amino acids located in the essential region of SEN1 has significant similarity to the yeast UPF1 gene product, which is involved in mRNA turnover, and the mouse Mov-10 gene product, whose function is unknown. The mutation that creates the temperature-sensitive sen1-1 allele is located within this 500-amino-acid region, and it causes a substitution for an amino acid that is conserved in all three proteins.

scholarly journals Reconstruction ofmreBExpression in Staphylococcus aureus via a Collection of New Integrative Plasmids

2014 ◽  
Vol 80 (13) ◽  
pp. 3868-3878 ◽  
Author(s):  
Ana Yepes ◽  
Gudrun Koch ◽  
Andrea Waldvogel ◽  
Juan-Carlos Garcia-Betancur ◽  
Daniel Lopez
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Cell Biology ◽  
Genetic Manipulation ◽  
Protein Localization ◽  
Antibiotic Resistance Genes ◽  
Wall Material ◽  
Content Type ◽  
Genetic Manipulations ◽  
Discrete Structures

ABSTRACTProtein localization has been traditionally explored in unicellular organisms, whose ease of genetic manipulation facilitates molecular characterization. The two rod-shaped bacterial modelsEscherichia coliandBacillus subtilishave been prominently used for this purpose and have displaced other bacteria whose challenges for genetic manipulation have complicated any study of cell biology. Among these bacteria is the spherical pathogenic bacteriumStaphylococcus aureus. In this report, we present a new molecular toolbox that facilitates gene deletion in staphylococci in a 1-step recombination process and additional vectors that facilitate the insertion of diverse reporter fusions into newly identified neutral loci of theS. aureuschromosome. Insertion of the reporters does not add any antibiotic resistance genes to the chromosomes of the resultant strains, thereby making them amenable for further genetic manipulations. We used this toolbox to reconstitute the expression ofmreBinS. aureus, a gene that encodes an actin-like cytoskeletal protein which is absent in coccal cells and is presumably lost during the course of speciation. We observed that inS. aureus, MreB is organized in discrete structures in association with the membrane, leading to an unusual redistribution of the cell wall material. The production of MreB also caused cell enlargement, but it did not revert staphylococcal shape. We present interactions of MreB with key staphylococcal cell wall-related proteins. This work facilitates the useS. aureusas a model system in exploring diverse aspects of cellular microbiology.

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