Abstract
The success of comparative cell biology for determining protein function relies on quality disruption techniques. Long-lived proteins, in postmitotic cells, are particularly difficult to eliminate. Moreover, cellular processes are notoriously adaptive; for example, neuronal synapses exhibit a high degree of plasticity. Ideally, protein disruption techniques should be both rapid and complete. Here, we describe knockoff, a generalizable method for the druggable control of membrane protein stability. We developed knockoff for neuronal use but show it also works in other cell types. Applying knockoff to synaptotagmin 1 (SYT1) results in acute disruption of this protein, resulting in loss of synchronous neurotransmitter release with a concomitant increase in the spontaneous release rate, measured optically. Thus, SYT1 is not only the proximal Ca2+ sensor for fast neurotransmitter release but also serves to clamp spontaneous release. Additionally, knockoff can be applied to protein domains as we show for another synaptic vesicle protein, synaptophysin 1.
Highlights
Protein function is frequently determined through removal or disruption of the protein of interest, followed by analysis of the resultant phenotype
We demonstrate the functionality and utility of knockoff by applying the system to synaptotagmin 1 (SYT1)
The findings reported here, using an optical approach, agree with a previous report concerning the fraction of synchronous release in wild type (WT) and Syt1 KO (S1KO) neurons (Nishiki and Augustine, 2004)
Summary
Protein function is frequently determined through removal or disruption of the protein of interest, followed by analysis of the resultant phenotype. That rely on protein tags, promise faster protein inactivation (usually in a KO or KD background) (Natsume and Kanemaki, 2017) Some of these methods include: chromophore/fluorophore assisted light inactivation (CALI/FALI) (Lin et al, 2013; Marek and Davis, 2002), auxininducible degron (AID) (Nishimura et al, 2009), and Trim-Away (Clift et al, 2017). Another approach, knocksideways, sequesters the protein of interest away from is site of action (Robinson et al, 2010). There remains an unmet need for a method to conduct non-invasive, rapid, and specific disruption of integral membrane
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