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PMID: 20200224 Published · ppublish English Journal Article Research Support, N.I.H., Extramural

Requirements for transitional endoplasmic reticulum site structure and function in Saccharomyces cerevisiae.

Molecular biology of the cell ·Vol. 21 ·No. 9 ·2010-05-01 ·Pages 1530-45

Shindiapina P, Barlowe C

Abstract

Secretory proteins are exported from the endoplasmic reticulum (ER) at specialized regions known as the transitional ER (tER). Coat protein complex II (COPII) proteins are enriched at tER sites, although the mechanisms underlying tER site assembly and maintenance are not understood. Here, we investigated the dynamic properties of tER sites in Saccharomyces cerevisiae and probed protein and lipid requirements for tER site structure and function. Thermosensitive sec12 and sec16 mutations caused a collapse of tER sites in a manner that depended on nascent secretory cargo. Continual fatty acid synthesis was required for ER export and for normal tER site structure, whereas inhibition of sterol and ceramide synthesis produced minor effects. An in vitro assay to monitor assembly of Sec23p-green fluorescent protein at tER sites was established to directly test requirements. tER sites remained active for approximately 10 min in vitro and depended on Sec12p function. Bulk phospholipids were also required for tER site structure and function in vitro, whereas depletion of phophatidylinositol selectively inhibited coat protein complex II (COPII) budding but not assembly of tER site structures. These results indicate that tER sites persist through relatively stringent treatments in which COPII budding was strongly inhibited. We propose that tER site structures are stable elements that are assembled on an underlying protein and lipid scaffold.

MeSH Terms
COP-Coated Vesicles/genetics,metabolism Cerulenin/pharmacology Cycloheximide/pharmacology Endoplasmic Reticulum/metabolism Fatty Acid Synthesis Inhibitors/pharmacology Fatty Acids/biosynthesis GTPase-Activating Proteins/genetics,metabolism Green Fluorescent Proteins/genetics,metabolism Guanine Nucleotide Exchange Factors/genetics,metabolism Immunoblotting Membrane Glycoproteins/genetics,metabolism Membrane Proteins/genetics,metabolism Microscopy, Confocal Microscopy, Fluorescence Mutation Nuclear Pore Complex Proteins/genetics,metabolism Phospholipases/metabolism Protein Synthesis Inhibitors/pharmacology Protein Transport/drug effects Recombinant Fusion Proteins/genetics,metabolism Saccharomyces cerevisiae/drug effects,genetics,metabolism Saccharomyces cerevisiae Proteins/genetics,metabolism
Chemicals
Fatty Acid Synthesis Inhibitors Fatty Acids GTPase-Activating Proteins Guanine Nucleotide Exchange Factors Membrane Glycoproteins Membrane Proteins Nuclear Pore Complex Proteins Protein Synthesis Inhibitors Recombinant Fusion Proteins SEC12 protein, S cerevisiae SEC13 protein, S cerevisiae SEC16 protein, S cerevisiae SEC23 protein, S cerevisiae Saccharomyces cerevisiae Proteins Green Fluorescent Proteins Cerulenin Cycloheximide Phospholipases
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Shindiapina Polina
Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA.
Barlowe Charles
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Article Info
Journal
Molecular biology of the cell
Abbr.
Mol Biol Cell
ISSN
1939-4586
DOI
10.1091/mbc.e09-07-0605
Published
2010-05-01
Epub
2010-00-03
Pages
1530-45
Language
English
Region
United States
NLM ID
9201390
PMCID
PMC2861612
Subset
IM
Grants
NIGMS NIH HHS · R37 GM052549 · United States
External Links
PubMed source DOI: 10.1091/mbc.e09-07-0605 PMC Full Text View journal details
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