Irradiation-induced protein inactivation reveals Golgi enzyme cycling to cell periphery

Department of Biological Sciences, Carnegie Mellon University, 4400 5th Avenue, Pittsburgh, PA 15213, USA.
Journal of Cell Science (Impact Factor: 5.43). 03/2012; 125(Pt 4):973-80. DOI: 10.1242/jcs.094441
Source: PubMed


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.

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    • "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. "
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    ABSTRACT: Rab proteins, small GTPases, are key regulators of mammalian Golgi apparatus organization. Based on the effect of Rab activation state, Rab proteins fall into two functional classes. In Class1, inactivation induces Golgi ribbon fragmentation and/or redistribution of Golgi enzymes to the Endoplasmic Reticulum, while overexpression of wild type or activation has little, if any, effect on Golgi ribbon organization. In Class 2, the reverse is true. We give emphasis to Rab6, the most abundant Golgi-associated Rab protein. Rab6 depletion in HeLa cells causes an increase in Golgi cisternal number, longer, more continuous cisternae, and a pronounced accumulation of vesicles; the effect of Rab6 on Golgi ribbon organization is probably through regulation of vesicle transport. In effector studies, motor proteins and their regulators are found to be key Rab6 effectors. A related Rab, Rab41, affects Golgi ribbon organization in a contrasting manner. The balance between minus- and plus-end directed motor recruitment may well be the major Rab-dependent factor in Golgi ribbon organization. Copyright © 2015 Elsevier Inc. All rights reserved.
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    • "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. "
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    ABSTRACT: Generating loss of protein function is a powerful investigatory tool particularly if carried out on a physiologically relevant timescale in a live-cell fluorescent imaging experiment. KillerRed mediated chromophore assisted light inactivation (CALI) uses genetic encoding for specificity and light for acute inactivation that can also be spatially restricted. This unit provides protocols for setting up and carrying out properly controlled KillerRed experiments during live-cell imaging of cultured cells. Curr. Protoc. Cytom. 69:12.35.1-12.35.10. © 2014 by John Wiley & Sons, Inc.
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