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Folding of a single domain protein entering the endoplasmic reticulum precedes disulfide formation.

ABSTRACT: The relationship between protein synthesis, folding, and disulfide formation within the endoplasmic reticulum (ER) is poorly understood. Previous studies have suggested that pre-existing disulfide links are absolutely required to allow protein folding and, conversely, that protein folding occurs prior to disulfide formation. To address the question of what happens first within the ER, that is, protein folding or disulfide formation, we studied folding events at the early stages of polypeptide chain translocation into the mammalian ER using stalled translation intermediates. Our results demonstrate that polypeptide folding can occur without complete domain translocation. Protein disulfide isomerase (PDI) interacts with these early intermediates, but disulfide formation does not occur unless the entire sequence of the protein domain is translocated. This is the first evidence that folding of the polypeptide chain precedes disulfide formation within a cellular context and highlights key differences between protein folding in the ER and refolding of purified proteins.

SUBMITTER: Robinson PJ

PROVIDER: S-EPMC5409466 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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Folding of a single domain protein entering the endoplasmic reticulum precedes disulfide formation.

Robinson Philip J PJ Pringle Marie Anne MA Woolhead Cheryl A CA Bulleid Neil J NJ

The Journal of biological chemistry 20170315 17

The relationship between protein synthesis, folding, and disulfide formation within the endoplasmic reticulum (ER) is poorly understood. Previous studies have suggested that pre-existing disulfide links are absolutely required to allow protein folding and, conversely, that protein folding occurs prior to disulfide formation. To address the question of what happens first within the ER, that is, protein folding or disulfide formation, we studied folding events at the early stages of polypeptide ch ...[more]

PMID: 28298446

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Project description:The protein secretory pathway must maintain homoeostasis while producing a wide assortment of proteins in different conditions. It is also used extensively to produce many useful proteins in biotechnology. As such, secretory pathway dysfunction can be highly detrimental to the cell, resulting in the molecular basis for many human diseases, and can drastically inhibit product titers in biochemical production. Because the secretory pathway is a highly-integrated, multi-organelle system, dysfunction can happen at many levels and dissecting the root cause can be challenging. To better understand some of these dysfunctions, we measured multiple systems-level states of the cell (physiology, transcriptome, metabolism) while secreting a small protein (insulin precursor) or a large protein (?-amylase). This was carried out in the presence and absence of HAC1, a key transcription factor in maintaining secretory homeostasis. Clear trends in cellular stress were apparent across multiple data resulting from our perturbations. In particular, processes involving (1) degradation of protein / recycling amino acids, (2) overall transcription/translation repression, and (3) oxidative stress. Apparent runaway oxidative radical production was explained by a thermodynamic model that we put forward for disulfide formation in the endoplasmic reticulum. This model predicts that balancing the relative rates of protein folding and disulfide bond formation are key to easing oxidative stress. These predictions have direct implications in how to engineer a broad range of recombinant proteins for secretion and provide potential hypotheses for the root causes of several secretory-associated diseases. Yeast strains were constructed that produce and secrete (a) IP or (b) ?-amylase and were compared to yeast strains containing (c) an empty vector in both wild-type and HAC1 deletion backgrounds. These strains are named WN (WT with empty vector), WI (WT secreting IP), WA (WT secreting ?-amylase), dN (?hac1 with empty vector), dI (?hac1 secreting IP), and dA (?hac1 secreting ?-amylase). Strains were characterized in batch fermentation and samples were taken in mid-exponential phase. Triplicate fermentations were carried out for each strain.

Dataset Information

Folding of a single domain protein entering the endoplasmic reticulum precedes disulfide formation.

Publications

Folding of a single domain protein entering the endoplasmic reticulum precedes disulfide formation.

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