Functional studies on components of the secretory pathway of Saccharomyces cerevisiae

Dissertation

Jaana Toikkanen

Research output: ThesisDissertationCollection of Articles

Abstract

SSO genes have been isolated as multicopy suppressors of sec1-1, a mutation in a late-acting SEC gene causing accumulation of post-Golgi vesicles at the restrictive temperature, and shown to encode the so-called target membrane SNAP receptors (t-SNAREs) in the plasma membrane of the yeast Saccharomyces cerevisiae.The Sso proteins are type II membrane proteins that are capable of posttranslational membrane insertion independent of signal recognition particles (SRPs).Thus, they might be inserted to the plasma membrane directly instead of entering the endoplasmic reticulum (ER) and being transported to their site of action along the biosynthetic pathway.The initial membrane insertion site of Sso2p in the secretory pathway was studied by transient expression of Sso2p in animal cells, which are superior to yeast for such morphological studies.Sso2p was shown to be first inserted into the ER and then transported to the plasma membrane via the secretory pathway.The Sso proteins are likely to be rate-limiting factors in the secretory machinery of S. cerevisiae because their overproduction enhanced by several fold the secretion of both a yeast protein, invertase, and a heterologous reporter protein, Bacillus a-amylase.Secretion to the periplasm was enhanced in the Sso2p-overproducing strain, consistent with a previously suggested role for plasma membrane t-SNAREs as anchors in targeting/fusion of the secretory vesicles to the plasma membrane.The secretion enhancement by overproduction of components of the secretory machinery offers a novel, more generally applicable approach to increase the rather modest secretory capacity of S. cerevisiae.The secretory pathway of yeast has been considered to contain other bottle necks e.g. in the ER and the Golgi complex.Interestingly, overexpression of a newly discovered gene, SEB1, shown to encode a novel subunit of an ER translocon, also resulted in enhanced secretion.SEB1 was cloned as a multicopy suppressor of sec15-1, a late-acting mutant gene.SEC15 shows an extensive pattern of genetic interactions with other late-acting SEC genes and is, thus, considered to have a central function in post-Golgi transport.It encodes a component of a multisubunit complex called the exocyst, which is implicated in targeting/fusion of the secretory vesicles to the plasma membrane as an effector of Sec4p.Surprisingly, the nonessential SEB1 gene encodes the evolutionarily conserved b subunit of the Sec61p complex, which functions in ER translocation.This unexpected finding prompted further studies on genetic interactions between SEB1 and the genes encoding exocyst subunits.Interestingly, SEB1, unlike its close homologue, SEB2, could suppress mutations in all of the exocyst genes.Furthermore, overproduction of the other two components of the ER translocon, Sec61p or Sss1p, could also suppress defects in many of the exocyst mutants.The double disruption seb1Dseb2D in combination with either of two mutant genes encoding exocyst components, sec10 or sec15, caused synthetic lethality, further strengthening the evidence for genetic interactions.The genetic interactions observed were rather specific for the two complexes, the ER translocon and the exocyst.In addition, overexpression of SEC1 or SSO2 could also suppress all the exocyst mutants except one and on the other hand all the genes encoding components of the Sec61p complex could suppress sec1-1 when overexpressed.These results suggest a closer interplay between the ER translocon, exocyst complex and plasma membrane t-SNARE than has previously been anticipated.
Original languageEnglish
QualificationDoctor Degree
Awarding Institution
  • University of Helsinki
Supervisors/Advisors
  • Keränen, Sirkka, Supervisor, External person
Award date11 Jun 1999
Place of PublicationEspoo
Publisher
Print ISBNs951-38-5381-0
Electronic ISBNs951-38-5382-9
Publication statusPublished - 1999
MoE publication typeG5 Doctoral dissertation (article)

Fingerprint

Secretory Pathway
Saccharomyces cerevisiae
Endoplasmic Reticulum
Cell Membrane
Genes
SNARE Proteins
Membranes
Yeasts
Secretory Vesicles
Signal Recognition Particle
Secretory Component
Gene Components
beta-Fructofuranosidase
Periplasm
Mutation
Proteins
Fungal Proteins
Biosynthetic Pathways
Golgi Apparatus
Amylases

Keywords

  • yeasts
  • Saccharomyces cerevisiae
  • genetic interactions
  • enhanced secretion
  • ER translocation
  • exocytosis
  • exocyst
  • heterologous proteins
  • SS02
  • SEB1
  • SEB2
  • SNAP receptors
  • plasma membrane

Cite this

Toikkanen, J. (1999). Functional studies on components of the secretory pathway of Saccharomyces cerevisiae: Dissertation. Espoo: VTT Technical Research Centre of Finland.
Toikkanen, Jaana. / Functional studies on components of the secretory pathway of Saccharomyces cerevisiae : Dissertation. Espoo : VTT Technical Research Centre of Finland, 1999. 95 p.
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abstract = "SSO genes have been isolated as multicopy suppressors of sec1-1, a mutation in a late-acting SEC gene causing accumulation of post-Golgi vesicles at the restrictive temperature, and shown to encode the so-called target membrane SNAP receptors (t-SNAREs) in the plasma membrane of the yeast Saccharomyces cerevisiae.The Sso proteins are type II membrane proteins that are capable of posttranslational membrane insertion independent of signal recognition particles (SRPs).Thus, they might be inserted to the plasma membrane directly instead of entering the endoplasmic reticulum (ER) and being transported to their site of action along the biosynthetic pathway.The initial membrane insertion site of Sso2p in the secretory pathway was studied by transient expression of Sso2p in animal cells, which are superior to yeast for such morphological studies.Sso2p was shown to be first inserted into the ER and then transported to the plasma membrane via the secretory pathway.The Sso proteins are likely to be rate-limiting factors in the secretory machinery of S. cerevisiae because their overproduction enhanced by several fold the secretion of both a yeast protein, invertase, and a heterologous reporter protein, Bacillus a-amylase.Secretion to the periplasm was enhanced in the Sso2p-overproducing strain, consistent with a previously suggested role for plasma membrane t-SNAREs as anchors in targeting/fusion of the secretory vesicles to the plasma membrane.The secretion enhancement by overproduction of components of the secretory machinery offers a novel, more generally applicable approach to increase the rather modest secretory capacity of S. cerevisiae.The secretory pathway of yeast has been considered to contain other bottle necks e.g. in the ER and the Golgi complex.Interestingly, overexpression of a newly discovered gene, SEB1, shown to encode a novel subunit of an ER translocon, also resulted in enhanced secretion.SEB1 was cloned as a multicopy suppressor of sec15-1, a late-acting mutant gene.SEC15 shows an extensive pattern of genetic interactions with other late-acting SEC genes and is, thus, considered to have a central function in post-Golgi transport.It encodes a component of a multisubunit complex called the exocyst, which is implicated in targeting/fusion of the secretory vesicles to the plasma membrane as an effector of Sec4p.Surprisingly, the nonessential SEB1 gene encodes the evolutionarily conserved b subunit of the Sec61p complex, which functions in ER translocation.This unexpected finding prompted further studies on genetic interactions between SEB1 and the genes encoding exocyst subunits.Interestingly, SEB1, unlike its close homologue, SEB2, could suppress mutations in all of the exocyst genes.Furthermore, overproduction of the other two components of the ER translocon, Sec61p or Sss1p, could also suppress defects in many of the exocyst mutants.The double disruption seb1Dseb2D in combination with either of two mutant genes encoding exocyst components, sec10 or sec15, caused synthetic lethality, further strengthening the evidence for genetic interactions.The genetic interactions observed were rather specific for the two complexes, the ER translocon and the exocyst.In addition, overexpression of SEC1 or SSO2 could also suppress all the exocyst mutants except one and on the other hand all the genes encoding components of the Sec61p complex could suppress sec1-1 when overexpressed.These results suggest a closer interplay between the ER translocon, exocyst complex and plasma membrane t-SNARE than has previously been anticipated.",
keywords = "yeasts, Saccharomyces cerevisiae, genetic interactions, enhanced secretion, ER translocation, exocytosis, exocyst, heterologous proteins, SS02, SEB1, SEB2, SNAP receptors, plasma membrane",
author = "Jaana Toikkanen",
year = "1999",
language = "English",
isbn = "951-38-5381-0",
series = "VTT Publications",
publisher = "VTT Technical Research Centre of Finland",
number = "389",
address = "Finland",
school = "University of Helsinki",

}

Toikkanen, J 1999, 'Functional studies on components of the secretory pathway of Saccharomyces cerevisiae: Dissertation', Doctor Degree, University of Helsinki, Espoo.

Functional studies on components of the secretory pathway of Saccharomyces cerevisiae : Dissertation. / Toikkanen, Jaana.

Espoo : VTT Technical Research Centre of Finland, 1999. 95 p.

Research output: ThesisDissertationCollection of Articles

TY - THES

T1 - Functional studies on components of the secretory pathway of Saccharomyces cerevisiae

T2 - Dissertation

AU - Toikkanen, Jaana

PY - 1999

Y1 - 1999

N2 - SSO genes have been isolated as multicopy suppressors of sec1-1, a mutation in a late-acting SEC gene causing accumulation of post-Golgi vesicles at the restrictive temperature, and shown to encode the so-called target membrane SNAP receptors (t-SNAREs) in the plasma membrane of the yeast Saccharomyces cerevisiae.The Sso proteins are type II membrane proteins that are capable of posttranslational membrane insertion independent of signal recognition particles (SRPs).Thus, they might be inserted to the plasma membrane directly instead of entering the endoplasmic reticulum (ER) and being transported to their site of action along the biosynthetic pathway.The initial membrane insertion site of Sso2p in the secretory pathway was studied by transient expression of Sso2p in animal cells, which are superior to yeast for such morphological studies.Sso2p was shown to be first inserted into the ER and then transported to the plasma membrane via the secretory pathway.The Sso proteins are likely to be rate-limiting factors in the secretory machinery of S. cerevisiae because their overproduction enhanced by several fold the secretion of both a yeast protein, invertase, and a heterologous reporter protein, Bacillus a-amylase.Secretion to the periplasm was enhanced in the Sso2p-overproducing strain, consistent with a previously suggested role for plasma membrane t-SNAREs as anchors in targeting/fusion of the secretory vesicles to the plasma membrane.The secretion enhancement by overproduction of components of the secretory machinery offers a novel, more generally applicable approach to increase the rather modest secretory capacity of S. cerevisiae.The secretory pathway of yeast has been considered to contain other bottle necks e.g. in the ER and the Golgi complex.Interestingly, overexpression of a newly discovered gene, SEB1, shown to encode a novel subunit of an ER translocon, also resulted in enhanced secretion.SEB1 was cloned as a multicopy suppressor of sec15-1, a late-acting mutant gene.SEC15 shows an extensive pattern of genetic interactions with other late-acting SEC genes and is, thus, considered to have a central function in post-Golgi transport.It encodes a component of a multisubunit complex called the exocyst, which is implicated in targeting/fusion of the secretory vesicles to the plasma membrane as an effector of Sec4p.Surprisingly, the nonessential SEB1 gene encodes the evolutionarily conserved b subunit of the Sec61p complex, which functions in ER translocation.This unexpected finding prompted further studies on genetic interactions between SEB1 and the genes encoding exocyst subunits.Interestingly, SEB1, unlike its close homologue, SEB2, could suppress mutations in all of the exocyst genes.Furthermore, overproduction of the other two components of the ER translocon, Sec61p or Sss1p, could also suppress defects in many of the exocyst mutants.The double disruption seb1Dseb2D in combination with either of two mutant genes encoding exocyst components, sec10 or sec15, caused synthetic lethality, further strengthening the evidence for genetic interactions.The genetic interactions observed were rather specific for the two complexes, the ER translocon and the exocyst.In addition, overexpression of SEC1 or SSO2 could also suppress all the exocyst mutants except one and on the other hand all the genes encoding components of the Sec61p complex could suppress sec1-1 when overexpressed.These results suggest a closer interplay between the ER translocon, exocyst complex and plasma membrane t-SNARE than has previously been anticipated.

AB - SSO genes have been isolated as multicopy suppressors of sec1-1, a mutation in a late-acting SEC gene causing accumulation of post-Golgi vesicles at the restrictive temperature, and shown to encode the so-called target membrane SNAP receptors (t-SNAREs) in the plasma membrane of the yeast Saccharomyces cerevisiae.The Sso proteins are type II membrane proteins that are capable of posttranslational membrane insertion independent of signal recognition particles (SRPs).Thus, they might be inserted to the plasma membrane directly instead of entering the endoplasmic reticulum (ER) and being transported to their site of action along the biosynthetic pathway.The initial membrane insertion site of Sso2p in the secretory pathway was studied by transient expression of Sso2p in animal cells, which are superior to yeast for such morphological studies.Sso2p was shown to be first inserted into the ER and then transported to the plasma membrane via the secretory pathway.The Sso proteins are likely to be rate-limiting factors in the secretory machinery of S. cerevisiae because their overproduction enhanced by several fold the secretion of both a yeast protein, invertase, and a heterologous reporter protein, Bacillus a-amylase.Secretion to the periplasm was enhanced in the Sso2p-overproducing strain, consistent with a previously suggested role for plasma membrane t-SNAREs as anchors in targeting/fusion of the secretory vesicles to the plasma membrane.The secretion enhancement by overproduction of components of the secretory machinery offers a novel, more generally applicable approach to increase the rather modest secretory capacity of S. cerevisiae.The secretory pathway of yeast has been considered to contain other bottle necks e.g. in the ER and the Golgi complex.Interestingly, overexpression of a newly discovered gene, SEB1, shown to encode a novel subunit of an ER translocon, also resulted in enhanced secretion.SEB1 was cloned as a multicopy suppressor of sec15-1, a late-acting mutant gene.SEC15 shows an extensive pattern of genetic interactions with other late-acting SEC genes and is, thus, considered to have a central function in post-Golgi transport.It encodes a component of a multisubunit complex called the exocyst, which is implicated in targeting/fusion of the secretory vesicles to the plasma membrane as an effector of Sec4p.Surprisingly, the nonessential SEB1 gene encodes the evolutionarily conserved b subunit of the Sec61p complex, which functions in ER translocation.This unexpected finding prompted further studies on genetic interactions between SEB1 and the genes encoding exocyst subunits.Interestingly, SEB1, unlike its close homologue, SEB2, could suppress mutations in all of the exocyst genes.Furthermore, overproduction of the other two components of the ER translocon, Sec61p or Sss1p, could also suppress defects in many of the exocyst mutants.The double disruption seb1Dseb2D in combination with either of two mutant genes encoding exocyst components, sec10 or sec15, caused synthetic lethality, further strengthening the evidence for genetic interactions.The genetic interactions observed were rather specific for the two complexes, the ER translocon and the exocyst.In addition, overexpression of SEC1 or SSO2 could also suppress all the exocyst mutants except one and on the other hand all the genes encoding components of the Sec61p complex could suppress sec1-1 when overexpressed.These results suggest a closer interplay between the ER translocon, exocyst complex and plasma membrane t-SNARE than has previously been anticipated.

KW - yeasts

KW - Saccharomyces cerevisiae

KW - genetic interactions

KW - enhanced secretion

KW - ER translocation

KW - exocytosis

KW - exocyst

KW - heterologous proteins

KW - SS02

KW - SEB1

KW - SEB2

KW - SNAP receptors

KW - plasma membrane

M3 - Dissertation

SN - 951-38-5381-0

T3 - VTT Publications

PB - VTT Technical Research Centre of Finland

CY - Espoo

ER -

Toikkanen J. Functional studies on components of the secretory pathway of Saccharomyces cerevisiae: Dissertation. Espoo: VTT Technical Research Centre of Finland, 1999. 95 p.