Academics

The Case of the Blocked ER-Golgi Pathway

In the recent Lindquist et al. (2006) study, they wanted to determine the effect of aSyn on the ER-Golgi pathway.  In order to accomplish this task, they took two approaches; one genetic and the other cellular.  Together these different pathways would converge to implicate aSyn in the blocking of ER-Golgi traffic and cell death.

aSyn was expressed in yeast and regulated with a galactose inducible promoter.  After aSyn expression, ER stress was measured and found to be increased for cells expressing aSyn-WT and further increased for the familial mutant aSyn-A53T.  Lindquist et al. (2006) hypothesized that aSyn was causing ER stress by blocking the function of endoplasmic reticulum associated degradation (ERAD).  As misfolded proteins accumulate in the ER, the ERAD process functions by retrotranslocating them back into the cytoplasm for proteasomal degradation (McCracken and Bdodsky 2006).  They found that out of two commonly misfolded proteins in the ER, CPY and Sec61-2p (both ERAD substrates), the rate of CPY degradation decreased even though proteasomal function was unaltered.  Interestingly, Caldwell et al. (2001) demonstrated that ERAD degradation of CPY required transport through the Golgi.

Because the failure of the ERAD translocation through the ER to the Golgi during aSyn expression may be an indicator of general pathway blockage, Lindquist et al. (2006) hypothesized that aSyn may be blocking ER-Golgi traffic.  To determine if this was the case, they followed two proteins, CPY and ALP, through the ER-Golgi circuit when aSyn was expressed.  Within three hours, ER-Golgi traffic was greatly reduced and at four hours nearly nonexistent.  Simultaneously, cell growth inhibition also occurred.  Thus, aSyn blocks ER-Golgi traffic (2006).

Following their cellular approach, Lindquist et al. (2006) initiated a genetics approach aimed at determining if genes that enhance ER-Golgi transport could reduce aSyn’s ability to block the pathway.  They identified the yeast protein Ypt1p as a promoter of traffic, and Gyp8P as a suppresser of traffic.  This finding led Lindquist et al. (2006) to hypothesize that over-expression of the Ypt1p (yeast) or Rab1 (mammalian) in a variety of models would rescue them from aSyn toxicity.

This final study yielded profound results that provided the strongest evidence, yet, that aSyn’s impairment of ER-Golgi traffic was the source for toxicity.  They overexpressed Rab1 along with aSyn in Drosophila melanogaster (fruit fly), C. elegans (worm), and mammalian dopaminergic neurons to determine if Rab1 would prevent aSyn toxicity by enhancing ER-Golgi traffic.  In all three models, the cells were rescued from death when overexpressing Rab1.

As a result of Ypt1p/Rab1 re-establishing ER-Golgi traffic, it was hypothesized that aSyn interacted at the ER-Golgi junction.  This was based on two lines of evidence; 1) CSP requires transport into the Golgi to be degraded and 2) Ypt1p/Rab1 functions within the ER-Golgi vesicular binding pathway.  Therefore, when Ypt1P/Rab1 is over-expressed, vesicular binding efficiency increases.

Returning to the Gosavi et al. (2002) and Lee et al. (2005) manuscripts, the Lindquist et al. (2006) data provides two established lines of evidence (i.e. detailed previously) supporting the Gosavi et al. (2002) conclusion that aSyn interacts with the ER-Golgi to yield toxicity.  Though aSyn is continuously being secreted through a Golgi-ER independent pathway (Lee et al., 2005), it is plausible that a defect in this excretory system may function to exacerbate toxicity, but not produce it.