ATPase activity of a yeast secretory glycoprotein allows ER exit during inactivation of COPII components Sec24p and Sec13p.

Proteins exit the endoplasmic reticulum (ER) in vesicles pinching off from the membrane at sites covered by the COPII coat, which consists of Sec23/24p and Sec13/31p. We have shown that the glycoprotein Hsp150 exits the ER in the absence of Sec13p or any member of the Sec24p family. The determinant responsible for this resides in the C-terminal domain of Hsp150 (CTD).

Unassisted translocation of large polypeptide domains across phospholipid bilayers.

Although transmembrane proteins generally require membrane-embedded machinery for integration, a few can insert spontaneously into liposomes. Previously, we established that the tail-anchored (TA) protein cytochrome b(5) (b5) can posttranslationally translocate 28 residues downstream to its transmembrane domain (TMD) across protein-free bilayers (Brambillasca, S., M.

Regulation and recovery of functions of Saccharomyces cerevisiae chaperone BiP/Kar2p after thermal insult.

We described earlier a novel mode of regulation of Hsp104, a cytosolic chaperone directly involved in the refolding of heat-denatured proteins, and designated it delayed upregulation, or DUR. When Saccharomyces cerevisiae cells grown at the physiological temperature of 24 degrees C, preconditioned at 37 degrees C, and treated briefly at 50 degrees C were shifted back to 24 degrees C, Hsp104 expression was strongly induced after 2.5 h of recovery and returned back to normal after 5 h.

Production of heterologous proteins in yeast with the aid of the Hsp150delta carrier.

Proper folding, and consequently exit from the endoplasmic reticulum (ER) and secretion of heterologous exocytic proteins in yeast can be rescued by fusing the proteins to certain yeast-derived polypeptides. Biologically active mammalian glycoproteins can be produced in Saccharomyces cerevisiae and Pichia pastoris by joining them to a fragment of a natural secretory glycoprotein of S. cerevisiae, Hsp150delta.

Transmembrane topogenesis of a tail-anchored protein is modulated by membrane lipid composition.

A large class of proteins with cytosolic functional domains is anchored to selected intracellular membranes by a single hydrophobic segment close to the C-terminus. Although such tail-anchored (TA) proteins are numerous, diverse, and functionally important, the mechanism of their transmembrane insertion and the basis of their membrane selectivity remain unclear. To address this problem, we have developed a highly specific, sensitive, and quantitative in vitro assay for the proper membrane-spanning topology of a model TA protein, cytochrome b5 (b5).

Endoplasmic reticulum exit of a secretory glycoprotein in the absence of sec24p family proteins in yeast.

Glycoproteins exit the endoplasmic reticulum (ER) of the yeast Saccharomyces cerevisiae in coat protein complex II (COPII) coated vesicles. The coat consists of the essential proteins Sec23p, Sec24p, Sec13p, Sec31p, Sar1p and Sec16p. Sec24p and its two nonessential homologues Sfb2p and Sfb3p have been suggested to serve in cargo selection.

Co-expression of two mammalian glycosyltransferases in the yeast cell wall allows synthesis of sLex.

Interactions between selectins and their oligosaccharide-decorated counter-receptors play an important role in the initiation of leukocyte extravasation in inflammation. L-selectin ligands are O-glycosylated with sulphated sialyl Lewis X epitopes (sulpho-sLex). Synthetic sLex oligosaccharides have been shown to inhibit adhesion of lymphocytes to endothelium at sites of inflammation.

Active and specific recruitment of a soluble cargo protein for endoplasmic reticulum exit in the absence of functional COPII component Sec24p.

Exit of proteins from the yeast endoplasmic reticulum (ER) is thought to occur in vesicles coated by four proteins, Sec13p, Sec31p, Sec23p and Sec24p, which assemble at ER exit sites to form the COPII coat. Sec13p may serve a structural function, whereas Sec24p has been suggested to operate in selection of cargo proteins into COPII vesicles. We showed recently that the soluble glycoprotein Hsp150 exited the ER in the absence of Sec13p function.

Upregulation of the Hsp104 chaperone at physiological temperature during recovery from thermal insult.

Thermal insult at 50 degrees C causes protein denaturation in yeast, but the cells survive if preconditioned at 37 degrees C. Survival depends on refolding of heat-denatured proteins. Refolding of cytoplasmic proteins requires Hsp104, the expression of which increases several-fold upon shift of the cells from physiological temperature 24 degrees C to 37 degrees C.

Activity of recycling Golgi mannosyltransferases in the yeast endoplasmic reticulum.

In yeast primary N- and O-glycans are attached to proteins in the endoplasmic reticulum (ER), and they are elongated in the Golgi. Thus, glycan extension by Golgi enzymes has been taken as evidence for arrival of a protein in the Golgi. Two alpha 1,6-mannosyltransferase activity-containing multiprotein complexes have been reported to recycle between the Golgi and the ER, but since resident ER proteins are not Golgi-modified, Golgi enzymes were not thought to function in the ER.

Validation of the Hsp150 polypeptide carrier and HSP150 promoter in expression of rat alpha2,3-sialyltransferase in yeasts.

Heterologous glycoproteins usually do not fold properly in yeast cells and fail to leave the endoplasmic reticulum. Here we show that the Hsp150Delta polypeptide carrier promoted proper folding and secretion of the catalytic ectodomain of rat alpha2,3-sialyltransferase (ST3Ne) in Pichia pastoris. The efficiency of the Hsp150Delta carrier in P.

Selective protein exit from yeast endoplasmic reticulum in absence of functional COPII coat component Sec13p.

Sec13p has been thought to be an essential component of the COPII coat, required for exit of proteins from the yeast endoplasmic reticulum (ER). We show herein that normal function of Sec13p was not required for ER exit of the Hsp150 glycoprotein. Hsp150 was secreted to the medium under restrictive conditions in a sec13-1 mutant.

Translocation of the C terminus of a tail-anchored protein across the endoplasmic reticulum membrane in yeast mutants defective in signal peptide-driven translocation.

C-tail-anchored proteins are defined by an N-terminal cytosolic domain followed by a transmembrane anchor close to the C terminus. Their extreme C-terminal polar residues are translocated across membranes by poorly understood post-translational mechanism(s). Here we have used the yeast system to study translocation of the C terminus of a tagged form of mammalian cytochrome b(5), carrying an N-glycosylation site in its C-terminal domain (b(5)-Nglyc).

GRACILE syndrome, a lethal metabolic disorder with iron overload, is caused by a point mutation in BCS1L.

GRACILE (growth retardation, aminoaciduria, cholestasis, iron overload, lactacidosis, and early death) syndrome is a recessively inherited lethal disease characterized by fetal growth retardation, lactic acidosis, aminoaciduria, cholestasis, and abnormalities in iron metabolism. We previously localized the causative gene to a 1.5-cM region on chromosome 2q33-37.