Michele L Forster

Harvard Medical School, Boston, Massachusetts, United States

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Publications (4)20.24 Total impact

  • Michele L Forster · James J Mahn · Billy Tsai
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    ABSTRACT: Protein-disulfide isomerase (PDI), an endoplasmic reticulum (ER)-resident protein, is primarily known as a catalyst of oxidative protein folding but also has a protein unfolding activity. We showed previously that PDI unfolds the cholera toxin A1 (CTA1) polypeptide to facilitate the ER-to-cytosol retrotranslocation of the toxin during intoxication. We now provide insight into the mechanism of this unfoldase activity. PDI includes two redox-active (a and a') and two redox-inactive (b and b') thioredoxin-like domains, a linker (x), and a C-terminal domain (c) arranged as abb'xa'c. Using recombinant PDI fragments, we show that binding of CTA1 by the continuous PDIbb'xa' fragment is necessary and sufficient to trigger unfolding. The specific linear arrangement of bb'xa' and the type a domain (a' versus a) C-terminal to bb'x are additional determinants of activity. These data suggest a general mechanism for the unfoldase activity of PDI: the concurrent and specific binding of bb'xa' to particular regions along the CTA1 molecule triggers its unfolding. Furthermore, we show the bb' domains of PDI are indispensable to the unfolding reaction, whereas the function of its a' domain can be substituted partially by the a' domain from ERp57 (abb'xa'c) or ERp72 (ca degrees abb'xa'), PDI-like proteins that do not unfold CTA1 normally. However, the bb' domains of PDI were insufficient to convert full-length ERp57 into an unfoldase because the a domain of ERp57 inhibited toxin binding. Thus, we propose that generating an unfoldase from thioredoxin-like domains requires the bb'(x) domains of PDI followed by an a' domain but not preceded by an inhibitory a domain.
    Journal of Biological Chemistry 04/2009; 284(19):13045-56. DOI:10.1074/jbc.M808352200 · 4.57 Impact Factor
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    Kaleena M Bernardi · Michele L Forster · Wayne I Lencer · Billy Tsai
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    ABSTRACT: Cholera toxin (CT) intoxicates cells by using its receptor-binding B subunit (CTB) to traffic from the plasma membrane to the endoplasmic reticulum (ER). In this compartment, the catalytic A1 subunit (CTA1) is unfolded by protein disulfide isomerase (PDI) and retro-translocated to the cytosol where it triggers a signaling cascade, leading to secretory diarrhea. How CT is targeted to the site of retro-translocation in the ER membrane to initiate translocation is unclear. Using a semipermeabilized-cell retro-translocation assay, we demonstrate that a dominant-negative Derlin-1-YFP fusion protein attenuates the ER-to-cytosol transport of CTA1. Derlin-1 interacts with CTB and the ER chaperone PDI as assessed by coimmunoprecipitation experiments. An in vitro membrane-binding assay showed that CTB stimulated the unfolded CTA1 chain to bind to the ER membrane. Moreover, intoxication of intact cells with CTB stabilized the degradation of a Derlin-1-dependent substrate, suggesting that CT uses the Derlin-1 pathway. These findings indicate that Derlin-1 facilitates the retro-translocation of CT. CTB may play a role in this process by targeting the holotoxin to Derlin-1, enabling the Derlin-1-bound PDI to unfold the A1 subunit and prepare it for transport.
    Molecular biology of the cell 04/2008; 19(3):877-84. DOI:10.1091/mbc.E07-08-0755 · 5.98 Impact Factor
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    ABSTRACT: Misfolded proteins in the endoplasmic reticulum (ER) are retained in the organelle or retrotranslocated to the cytosol for proteasomal degradation. ER chaperones that guide these opposing processes are largely unknown. We developed a semipermeabilized cell system to study the retrotranslocation of cholera toxin (CT), a toxic agent that crosses the ER membrane to reach the cytosol during intoxication. We found that protein disulfide isomerase (PDI) facilitates CT retrotranslocation, whereas ERp72, a PDI-like protein, mediates its ER retention. In vitro analysis revealed that PDI and ERp72 alter CT's conformation in a manner consistent with their roles in retrotranslocation and ER retention. Moreover, we found that PDI's and ERp72's opposing functions operate on endogenous ER misfolded proteins. Thus, our data identify PDI family proteins that play opposing roles in ER quality control and establish an assay to further delineate the mechanism of CT retrotranslocation.
    The Journal of Cell Biology 07/2006; 173(6):853-9. DOI:10.1083/jcb.200602046 · 9.69 Impact Factor
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    Michele L. Forster
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    ABSTRACT: The endoplasmic reticulum (ER) quality control system retains nascent polypeptides in the organelle until they are folded properly and mediates the ER-to-cytosol transport (i.e., retrotranslocation) of misfolded proteins for proteasomal degradation. In mammalian cells, the retrotranslocation machinery is exploited by the A1 subunit of cholera toxin (CTA1) for its transport into the cytosol where it induces toxicity. Using a semi-permeabilized cell system characterized here, we found the ER factor protein disulfide isomerase (PDI) facilitates CTA1 retrotranslocation consistent with its previously demonstrated ability to unfold the toxin in vitro. In contrast, the PDI family protein ERp72 retains CTA1 in the ER and stabilizes a folded conformation of the toxin. PDI and ERp72’s opposing functions also operate on proteins misfolded in the ER. Thus, we identified PDI family proteins that play opposing roles during ER quality control and established an assay to study CTA1 retrotranslocation. Because PDI’s unfoldase activity correlates with retrotranslocation, we investigated the mechanism of this activity. PDI comprises two redox active (a and a’) and two redox inactive (b and b’) thioredoxin-like domains, a linker (x), and a C-terminal domain (c) arranged abb’xa’c. Using recombinant PDI fragments, we show that binding of CTA1 by PDI’s bb’xa’ domains is necessary and sufficient to trigger unfolding. The linear arrangement of bb’xa’ and type of a domain (a’ versus a) C-terminal to bb’x are additional determinants of activity. These data suggest a general mechanism for PDI’s unfoldase activity: the concurrent and specific binding of bb’xa’ to particular regions along the CTA1 molecule triggers its unfolding. Furthermore, we show that PDI’s bb’ domains are indispensable to the unfolding reaction, whereas the function of its a’ domain can be substituted partially by the a’ domain from ERp57 (abb’xa’c) or ERp72 (caºabb’xa’), PDI family proteins that do not unfold CTA1 normally. However, PDI’s bb’ domains were insufficient to convert full-length ERp57 into an unfoldase because ERp57’s a domain inhibited toxin binding. Thus, we propose that generating an unfoldase from thioredoxin-like domains requires PDI’s bb’(x) domains followed by an a’ domain but not preceded by an a domain that can inhibit binding. Ph.D. Cell and Developmental Biology University of Michigan, Horace H. Rackham School of Graduate Studies http://deepblue.lib.umich.edu/bitstream/2027.42/62334/1/mlfor_1.pdf

Publication Stats

170 Citations
20.24 Total Impact Points

Institutions

  • 2008
    • Harvard Medical School
      Boston, Massachusetts, United States
  • 2006
    • Concordia University–Ann Arbor
      Ann Arbor, Michigan, United States