A novel and simple method of production and biophysical characterization of a mini‐membrane protein, Ost4p: A Subunit of yeast oligosaccharyl transferase

Kumar, Amit; Ward, Priscilla; Katre, Uma V.; Mohanty, Smita

doi:10.1002/bip.22028

Cited by 8 publications

(1 citation statement)

References 31 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Ost4 stabilizes Stt3 and helps in the recruitment of Ost3/Ost6 as well [16,30,31]. Recombinant Ost4 and Ost4V23D mutant proteins have been successfully expressed, purified, and reconstituted in detergent for structure-function studies [73,74]. Ost3, a subunit that is homologous to Ost6, contains four transmembrane helices.…”

Section: Non-catalytic Subunitsmentioning

confidence: 99%

Structural Insight into the Mechanism of N-Linked Glycosylation by Oligosaccharyltransferase

2020

Self Cite

View full text Add to dashboard Cite

Asparagine-linked glycosylation, also known as N-linked glycosylation is an essential and highly conserved post-translational protein modification that occurs in all three domains of life. This modification is essential for specific molecular recognition, protein folding, sorting in the endoplasmic reticulum, cell–cell communication, and stability. Defects in N-linked glycosylation results in a class of inherited diseases known as congenital disorders of glycosylation (CDG). N-linked glycosylation occurs in the endoplasmic reticulum (ER) lumen by a membrane associated enzyme complex called the oligosaccharyltransferase (OST). In the central step of this reaction, an oligosaccharide group is transferred from a lipid-linked dolichol pyrophosphate donor to the acceptor substrate, the side chain of a specific asparagine residue of a newly synthesized protein. The prokaryotic OST enzyme consists of a single polypeptide chain, also known as single subunit OST or ssOST. In contrast, the eukaryotic OST is a complex of multiple non-identical subunits. In this review, we will discuss the biochemical and structural characterization of the prokaryotic, yeast, and mammalian OST enzymes. This review explains the most recent high-resolution structures of OST determined thus far and the mechanistic implication of N-linked glycosylation throughout all domains of life. It has been shown that the ssOST enzyme, AglB protein of the archaeon Archaeoglobus fulgidus, and the PglB protein of the bacterium Campylobactor lari are structurally and functionally similar to the catalytic Stt3 subunit of the eukaryotic OST enzyme complex. Yeast OST enzyme complex contains a single Stt3 subunit, whereas the human OST complex is formed with either STT3A or STT3B, two paralogues of Stt3. Both human OST complexes, OST-A (with STT3A) and OST-B (containing STT3B), are involved in the N-linked glycosylation of proteins in the ER. The cryo-EM structures of both human OST-A and OST-B complexes were reported recently. An acceptor peptide and a donor substrate (dolichylphosphate) were observed to be bound to the OST-B complex whereas only dolichylphosphate was bound to the OST-A complex suggesting disparate affinities of two OST complexes for the acceptor substrates. However, we still lack an understanding of the independent role of each eukaryotic OST subunit in N-linked glycosylation or in the stabilization of the enzyme complex. Discerning the role of each subunit through structure and function studies will potentially reveal the mechanistic details of N-linked glycosylation in higher organisms. Thus, getting an insight into the requirement of multiple non-identical subunits in the N-linked glycosylation process in eukaryotes poses an important future goal.

show abstract

Section: Non-catalytic Subunitsmentioning

confidence: 99%

Structural Insight into the Mechanism of N-Linked Glycosylation by Oligosaccharyltransferase

2020

Self Cite

View full text Add to dashboard Cite

show abstract

Membrane Protein Production and Purification from Escherichia coli and Sf9 Insect Cells

Liu

Pavić

Farley

et al. 2020

Methods in Molecular Biology

View full text Add to dashboard Cite

A major obstacle for studying membrane proteins by biophysical techniques is the difficulty to produce sufficient amount of materials for functional and structural studies. To overexpress the target membrane protein heterologously, especially a eukaryotic protein, a key step is to find the optimal host expression system and perform subsequent expression optimization. In this chapter, we describe protocols for screening membrane protein production using bacteria and insect cells, solubilization screening, large-scale production as well as commonly used affinity chromatography purification methods. We discuss general optimisation conditions such as promoters, tags and describe current techniques that can be used in any laboratory without specialised expensive equipment. Especially for insect cells, GFP-fusions are particularly useful for localisation and in-gel fluorescence detection of the proteins on SDS-PAGE. We give detailed protocols that can be used to screen the best expression and purification conditions for membrane protein study.

show abstract

Reconstitution and resonance assignments of yeast OST subunit Ost4 and its critical mutant Ost4V23D in liposomes by solid-state NMR

Chaudhary,

Struppe,

Moktan

et al. 2024

J Biomol NMR

View full text Add to dashboard Cite

A novel and simple method of production and biophysical characterization of a mini‐membrane protein, Ost4p: A Subunit of yeast oligosaccharyl transferase

Cited by 8 publications

References 31 publications

Structural Insight into the Mechanism of N-Linked Glycosylation by Oligosaccharyltransferase

Structural Insight into the Mechanism of N-Linked Glycosylation by Oligosaccharyltransferase

Membrane Protein Production and Purification from Escherichia coli and Sf9 Insect Cells

Reconstitution and resonance assignments of yeast OST subunit Ost4 and its critical mutant Ost4V23D in liposomes by solid-state NMR

Contact Info

Product

Resources

About