Analysis of Protein Turnover by Quantitative SNAP‐Based Pulse‐Chase Imaging

Abstract

Assessment of protein dynamics in living cells is crucial for understanding their biological properties and functions. The SNAP‐tag, a self labeling suicide enzyme, presents a tool with unique features that can be adopted for determining protein dynamics in living cells. Here we present detailed protocols for the use of SNAP in fluorescent pulse‐chase and quench‐chase‐pulse experiments. These time‐slicing methods provide powerful tools to assay and quantify the fate and turnover rate of proteins of different ages. We cover advantages and pitfalls of SNAP‐tagging in fixed‐ and live‐cell studies and evaluate the recently developed fast‐acting SNAPf variant. In addition, to facilitate the analysis of protein turnover datasets, we present an automated algorithm for spot recognition and quantification. Curr. Protoc. Cell Biol. 55:8.8.1‐8.8.34. © 2012 by John Wiley & Sons, Inc.

Activity-Dependent Degradation of Synaptic Vesicle Proteins Requires Rab35 and the ESCRT Pathway

“…GTP-bound Rabs in turn recruit effectors that catalyze downstream trafficking events (Stenmark, 2009). Among the Ͼ60 Rabs in mammalian brain, at least 30 are reported to associate with SV pools (Takamori et al, 2006;Pavlos and Jahn, 2011), but only a few of these have well-established roles in the SV cycle (Schlüter et al, 1999(Schlüter et al, , 2004Mahoney et al, 2006;Yu et al, 2008;Pavlos et al, 2010;Pavlos and Jahn, 2011). Several other Rabs are implicated in the endosomal sorting of SV proteins (Wucherpfennig et al, 2003;Rizzoli et al, 2006;Takamori et al, 2006;Hoopmann et al, 2010;Uytterhoeven et al, 2011).…”

“…The ESCRT system and homotypic fusion and vacuole proteinsorting complex were identified through genetic interactions as downstream components of this pathway (Uytterhoeven et al, 2011;Fernandes et al, 2014). However, other SVassociated Rabs (i.e.,5,11,26) are implicated in macroautophagy (Ao et al, 2014;Binotti et al, 2015), another lysosomal degradative pathway that may also be important for SV protein turnover. Indeed, macroautophagy has been shown to mediate the degradation of SVs under certain conditions (Hernandez et al, 2012).…”

Roles for Trafficking and O-Linked Glycosylation in the Turnover of Model Cell Surface Proteins

“…BG can be conjugated to a variety of fluorophores and other labels (30), making it possible to tag and track the protein of interest directly by a variety of methods such as fluorescence microscopy, gel electrophoresis, and in vivo imaging (31)(32)(33). The SNAP-tag has been demonstrated not to affect the function of a large number of fusion proteins (34,35) and is an optimal approach for pulse-chase labeling experiments (34,36). The covalent bond between BG and the SNAP-tag, however, makes fluorescence studies of endocytosis difficult because the probe cannot be removed from labeled proteins on the cell surface, concealing the intracellular endocytosed pool.…”

Breaking the color barrier – a multi-selective antibody reporter offers innovative strategies of fluorescence detection

“…3). Application-wise, a tunable signal detection reporter offers opportunities for advanced protein discovery strategies involving protein turnover at the cellular membrane, pulse-chase of vesicular traffic or post-cell-labeling assays that require the removal of signal (Bodor et al, 2012;Fuchs et al, 2010;Giepmans et al, 2006;Lippincott-Schwartz and Patterson, 2003;Mizukami et al, 2012). Furthermore, in order to increasingly accelerate the off-rates of signal detection using multiselective FAPs, the future focus will be placed on isolating weakaffinity fluorogens.…”