Improperly folded green fluorescent protein is secreted via a non-classical pathway

Abstract

The green fluorescent protein is a cytosolic protein frequently used as a molecular tag to study protein localization in intact cells. We discovered that this protein is secreted into the medium by several but not all cell lines through a non-classical secretory pathway that is insensitive to brefeldin A. Green fluorescent protein is secreted efficiently by Chinese hamster ovary cells, with 60% of synthesized proteins secreted over 8 hours. This pathway is sensitive to changes in temperature but not to factors in serum or chemicals known to affect other non-classical protein secretion pathways. Fluorescence is observed in cells expressing green fluorescent protein, indicating that some of the protein must be fully folded in the cytosol. However, secreted green fluorescent protein is not fluorescent and therefore not folded properly. Furthermore, cellular fluorescence does not change over 6 hours whereas a significant proportion of green fluorescent protein is secreted. Thus, nascent green fluorescent protein either is folded correctly or incorrectly, and the improperly folded molecules can be exported. Non-classical secretion might be a route by which cells remove an excess of improperly folded, cytosolic proteins.

Secretion and Immunogenicity of the Meningioma-Associated Antigen TXNDC16

“…After confirmation of the GFP translocation, we analyzed HEK293 cells transiently expressing the above-mentioned GFP variants. Wild-type GFP served as control and was detected almost exclusively intracellularly, although a small amount was found in the supernatant, presumably an artifact of GFP overexpression as previously described by Tanudji et al (30). SP-TX-GFP-TX-Cterm was exclusively detected in supernatant, which was similar to SP-TX-GFP and proves that TXNDC16 C terminus does not have any influence on ER retention (Fig.…”

Cysteine Oxidation Targets Peroxiredoxins 1 and 2 for Exosomal Release through a Novel Mechanism of Redox-Dependent Secretion

“…This is supported by experiments showing that, when expressed as a GFP-fusion protein, the C51S mutant is present in large aggregates in the cytoplasm, while the wild-type protein is present as uniformly distributed punctate staining in the cytoplasm. Secretion of misfolded or overexpressed proteins is a phenomenon previously described for various proteins and is thought to represent a means by which the cytoplasm can be cleared of aggregated or accumulated protein (24,25). As there does not appear to be any difference in the extent of expression for the wild-type and C51S mutant, it seems more likely that the release of the monomer in the case of the C51S mutant is due to the lack of dimerization which seems to cause aggregation within the cells.…”

Aldehyde dehydrogenase 1A1 and gelsolin identified as novel invasion-modulating factors in conditioned medium of pancreatic cancer cells

“…In addition, recent studies have shown that the non-classical secretory pathway works independently of the ER-Golgi network; the secreted proteins do not enter the ER and have not been glycosylated [15]. Nonclassical secretion by cell lines of the cytosolic green fluorescent protein (GFP) was shown experimentally [16]; export was not hampered by inhibitors of the classical secretory pathway, such as monensin and brefeldin A. Martin et al [17] also identified many intracellular proteins from both the cytoplasmic and nuclear compartments from proteomics analysis of medium conditioned by the prostate cell line, LNCaP. During cell culture in serum free conditions, some cells will die, resulting in the release of intracellular proteins into the media.…”