Acute inhibition is a powerful technique to test proteins for direct roles and order their activities in a pathway, but as a general gene-based strategy, it is mostly unavailable in mammalian systems. As a consequence, the precise roles of proteins in membrane trafficking have been difficult to assess in vivo. Here we used a strategy based on a genetically encoded fluorescent protein that generates highly localized and damaging reactive oxygen species to rapidly inactivate exit from the endoplasmic reticulum (ER) during live-cell imaging and address the long-standing question of whether the integrity of the Golgi complex depends on constant input from the ER. Light-induced blockade of ER exit immediately perturbed Golgi membranes, and surprisingly, revealed that cis-Golgi-resident proteins continuously cycle to peripheral ER-Golgi intermediate compartment (ERGIC) membranes and depend on ER exit for their return to the Golgi. These experiments demonstrate that ER exit and extensive cycling of cis-Golgi components to the cell periphery sustain the mammalian Golgi complex.
"Rab1, as suggested earlier, might be an example of this. Rab1 is required for the organization of ER-Golgi intermediate compartment (Jarvela and Linstedt, 2012). In the absence of the intermediate compartment, machinery recycling should be profoundly affected. "
International review of cell and molecular biology 02/2015; 315:1-22. DOI:10.1016/bs.ircmb.2014.12.002 · 3.42 Impact Factor
"This ensures that all copies of the protein of interest are light sensitive and allows a test of the constructs' ability to function prior to its inactivation. Significantly, however, if the target protein forms dimers or multimers, it is possible to inactivate endogenous proteins in trans, meaning that the transfected construct interacts with its endogenous partner and both are inactivated (Jarvela and Linstedt, 2012, 2014). (4) KillerRed should be paired with fluorescent reporters responding to other wavelengths to read out the physiological activity of interest. "
[Show abstract][Hide abstract] ABSTRACT: Bacterial AB5 toxins are a clinically relevant class of exotoxins that include several well-known members such as Shiga, cholera, and pertussis toxins. Infections with toxin-producing bacteria cause devastating human diseases that affect millions of individuals each year and have no definitive medical treatment. The molecular targets of AB5 toxins reside in the cytosol of infected cells, and the toxins reach the cytosol by trafficking through the retrograde membrane transport pathway that avoids degradative late endosomes and lysosomes. Focusing on Shiga toxin as the archetype member, we review recent advances in understanding the molecular mechanisms involved in the retrograde trafficking of AB5 toxins and highlight how these basic science advances are leading to the development of a promising new therapeutic approach based on inhibiting toxin transport.
Journal of Molecular Medicine 05/2013; 91(10). DOI:10.1007/s00109-013-1048-7 · 5.11 Impact Factor
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