Export is the default pathway for soluble unfolded polypeptides that accumulate during expression in Escherichia coli

Journal Article · Thu Sep 01 04:00:00 EDT 2011 · Protein Expression and Purification

DOI:https://doi.org/10.1016/j.pep.2011.03.011· OSTI ID:1040554

Scotto-Lavino, E; Freimuth, P; Bai, M; Zhang, Y -B

Several E. coli endogenous, cytoplasmic proteins that are known clients of the chaperonin GroEL were overexpressed to examine the fate of accumulated unfolded polypeptides. Substantial fractions of about half of the proteins formed insoluble aggregates, consistent with the hypothesis that these proteins were produced at rates or in amounts that exceeded the protein-folding capacity of GroEL. In addition, large fractions of three overexpressed GroEL client proteins were localized in an extra-cytoplasmic, osmotically-sensitive compartment, suggesting they had initially accumulated in the cytoplasm as soluble unfolded polypeptides and thus were able to access a protein export pathway. Consistent with this model, an intrinsically unfoldable, hydrophilic, non-secretory polypeptide was quantitatively exported from the E. coli cytoplasm into an osmotically-sensitive compartment. Our results support the conclusion that a soluble, unfolded conformation alone may be sufficient to direct non-secretory polypeptides into a protein export pathway for signal peptide-independent translocation across the inner membrane, and that export rather than degradation by cytoplasmic proteases is the preferred fate for newly-synthesized, soluble, unfolded polypeptides that accumulate in the cytoplasm. The stable folded conformation of exported GroEL client proteins further suggests that the requirement for GroEL may be conditional on protein folding in the molecularly-crowded environment of the cytoplasm.

• Research Organization: BROOKHAVEN NATIONAL LABORATORY (BNL)
• Sponsoring Organization: LABORATORY-DIRECTED RESEARCH AND DEVELOPMENT
• DOE Contract Number: AC02-98CH10886
• OSTI ID: 1040554
• Report Number(s): BNL--95384-2011-JA; YN0100000
• Journal Information: Protein Expression and Purification, Journal Name: Protein Expression and Purification Journal Issue: 1 Vol. 79; ISSN 1046-5928
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
60 APPLIED LIFE SCIENCES
CAPACITY
CYTOPLASM
ESCHERICHIA COLI
EXPORTS
GroEL
HEAT-SHOCK PROTEINS
HYPOTHESIS
POLYPEPTIDES
PROTEINS
TRANSLOCATION
export
molecular chaperones
overexpression
protein folding