scholarly journals Shr3p Mediates Specific COPII Coatomer–Cargo Interactions Required for the Packaging of Amino Acid Permeases Into ER-derived Transport Vesicles

1999 ◽  
Vol 10 (11) ◽  
pp. 3549-3565 ◽  
Author(s):  
C. Fredrik Gilstring ◽  
Monika Melin-Larsson ◽  
Per O. Ljungdahl
Keyword(s):  
Amino Acid ◽  
Temperature Sensitive ◽  
Amino Acid Permease ◽  
Terminal Domain ◽  
Carboxyl Terminal Domain ◽  
Transport Vesicles ◽  
Transport Vesicle ◽  
Amino Acid Permeases ◽  
Carboxyl Terminal ◽  
Membrane Spanning

The SHR3 gene of Saccharomyces cerevisiae encodes an integral membrane component of the endoplasmic reticulum (ER) with four membrane-spanning segments and a hydrophilic, cytoplasmically oriented carboxyl-terminal domain. Mutations in SHR3 specifically impede the transport of all 18 members of the amino acid permease (aap) gene family away from the ER. Shr3p does not itself exit the ER. Aaps fully integrate into the ER membrane and fold properly independently of Shr3p. Shr3p physically associates with the general aap Gap1p but not Sec61p, Gal2p, or Pma1p in a complex that can be purified fromN-dodecylmaltoside-solubilized membranes. Pulse–chase experiments indicate that the Shr3p–Gap1p association is transient, a reflection of the exit of Gap1p from the ER. The ER-derived vesicle COPII coatomer components Sec13p, Sec23p, Sec24p, and Sec31p but not Sar1p bind Shr3p via interactions with its carboxyl-terminal domain. The mutant shr3-23p, a nonfunctional membrane-associated protein, is unable to associate with aaps but retains the capacity to bind COPII components. The overexpression of either Shr3p or shr3-23p partially suppresses the temperature-sensitive sec12-1 allele. These results are consistent with a model in which Shr3p acts as a packaging chaperone that initiates ER-derived transport vesicle formation in the proximity of aaps by facilitating the membrane association and assembly of COPII coatomer components.

scholarly journals Amino acid permeases require COPII components and the ER resident membrane protein Shr3p for packaging into transport vesicles in vitro.

1996 ◽  
Vol 135 (3) ◽  
pp. 585-595 ◽  
Author(s):  
M J Kuehn ◽  
R Schekman ◽  
P O Ljungdahl
Keyword(s):  
Plasma Membrane ◽  
Amino Acid ◽  
Membrane Protein ◽  
Integral Membrane Protein ◽  
Plasma Membrane Atpase ◽  
Amino Acid Permease ◽  
Transport Vesicles ◽  
Amino Acid Permeases ◽  
Histidine Permease

In S. cerevisiae lacking SHR3, amino acid permeases specifically accumulate in membranes of the endoplasmic reticulum (ER) and fail to be transported to the plasma membrane. We examined the requirements of transport of the permeases from the ER to the Golgi in vitro. Addition of soluble COPII components (Sec23/24p, Sec13/31p, and Sar1p) to yeast membrane preparations generated vesicles containing the general amino acid permease. Gap1p, and the histidine permease, Hip1p. Shr3p was required for the packaging of Gap1p and Hip1p but was not itself incorporated into transport vesicles. In contrast, the packaging of the plasma membrane ATPase, Pma1p, and the soluble yeast pheromone precursor, glycosylated pro alpha factor, was independent of Shr3p. In addition, we show that integral membrane and soluble cargo colocalize in transport vesicles, indicating that different types of cargo are not segregated at an early step in secretion. Our data suggest that specific ancillary proteins in the ER membrane recruit subsets of integral membrane protein cargo into COPII transport vesicles.

scholarly journals The SHR3 homologue from S. pombe demonstrates a conserved function of ER packaging chaperones

2000 ◽  
Vol 113 (23) ◽  
pp. 4351-4362 ◽  
Author(s):  
P. Martinez ◽  
P.O. Ljungdahl
Keyword(s):  
Endoplasmic Reticulum ◽  
Amino Acid ◽  
Amino Acid Uptake ◽  
Acid Uptake ◽  
Dependent Manner ◽  
Transport Vesicles ◽  
Transport Vesicle ◽  
Amino Acid Permeases ◽  
Mutant Phenotypes ◽  
High Degree

In Saccharomyces cerevisiae cells lacking SHR3, amino acid permeases do not enter into COPII transport vesicles and specifically accumulate in the membrane of the endoplasmic reticulum. Shr3p functions as a packaging chaperone to prime transport vesicle formation in the proximity of amino acid permeases. A genetic screen was developed that enabled the Schizosaccharomyces pombe SHR3 functional homologue, designated psh3(+) (pombe SHR3), to be cloned. The psh3(+) gene encodes a protein of 215 amino acids, which shares a high degree of structural and functional similarity with Shr3p. The heterologous expression of psh3(+) complements many, but not all, shr3 null mutant phenotypes in S. cerevisiae in a temperature-dependent manner. Psh3p is localised to the endoplasmic reticulum of S. pombe cells, and strains lacking the psh3(+)gene exhibit decreased rates of amino acid uptake due to reduced levels of functional permeases in the plasma membrane. No packaging chaperones, or proteins exhibiting homology with packaging chaperones, have so far been identified in other eukayotic organisms. The findings reported here are the first to establish that specific packaging chaperones exist in divergent organisms, and demonstrate a conserved function of packaging chaperones in facilitating the export of large polytopic membrane proteins from the endoplasmic reticulum.

scholarly journals Control of Amino Acid Permease Sorting in the Late Secretory Pathway of Saccharomyces cerevisiae by SEC13, LST4, LST7 and LST78

Genetics ◽  
1997 ◽  
Vol 147 (4) ◽  
pp. 1569-1584 ◽  
Author(s):  
Kevin J Roberg ◽  
Stephen Bickel ◽  
Neil Rowley ◽  
Chris A Kaiser
Keyword(s):  
Amino Acid ◽  
Cell Surface ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Secretory Vesicle ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Amino Acid Permease ◽  
New Genes ◽  
Amino Acid Permeases

Abstract The SEC13 gene was originally identified by temperature-sensitive mutations that block all protein transport from the ER to the Golgi. We have found that at a permissive temperature for growth, the sec13-1 mutation selectively blocks transport of the nitrogen-regulated amino acid permease, Gaplp, from the Golgi to the plasma membrane, but does not affect the activity of constitutive permeases such as Hip1p, Can1p, or Lyp1p. Different alleles of SEC13 exhibit different relative effects on protein transport from the ER to the Golgi, or on Gap1p activity, indicating distinct requirements for SEC13 function at two different steps in the secretory pathway. Three new genes, LST4, LST7, and LSTB, were identified that are also required for amino acid permease transport from the Golgi to the cell surface. Mutations in LST4 and LST7 reduce the activity of the nitrogen-regulated permeases Gap1p and Put4p, whereas mutations in LST8 impair the activities of a broader set of amino acid permeases. The LST8 gene encodes a protein composed of WD-repeats and has a close human homologue. The LST7 gene encodes a novel protein. Together, these data indicate that SEC13, LST4, LST7, and LST8 function in the regulated delivery of Gap1p to the cell surface, perhaps as components of a post-Golgi secretory-vesicle coat.

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