Incomplete proteasomal degradation of green fluorescent proteins in the context of tandem fluorescent protein timers

Abstract

Tandem fluorescent protein timers (tFTs) report on protein age through time-dependent change in color, which can be exploited to study protein turnover and trafficking. Each tFT, composed of two fluorescent proteins (FPs) that differ in maturation kinetics, is suited to follow protein dynamics within a specific time range determined by the maturation rates of both FPs. So far, tFTs have been constructed by combining slower-maturing red fluorescent proteins (redFPs) with the faster-maturing superfolder green fluorescent protein (sfGFP). Toward a comprehensive characterization of tFTs, we compare here tFTs composed of different faster-maturing green fluorescent proteins (greenFPs) while keeping the slower-maturing redFP constant (mCherry). Our results indicate that the greenFP maturation kinetics influences the time range of a tFT. Moreover, we observe that commonly used greenFPs can partially withstand proteasomal degradation due to the stability of the FP fold, which results in accumulation of tFT fragments in the cell. Depending on the order of FPs in the timer, incomplete proteasomal degradation either shifts the time range of the tFT toward slower time scales or precludes its use for measurements of protein turnover. We identify greenFPs that are efficiently degraded by the proteasome and provide simple guidelines for the design of new tFTs. INTRODUCTIONFluorescent proteins (FPs) become fluorescent only upon correct folding of the polypeptide and formation of the fluorophore through a series of chemical reactions within the β-barrel fold. This maturation process takes place on time scales from minutes to hours, depending on the FP and the environment (Tsien, 1998;Shaner et al., 2005). The time delay between protein synthesis and completion of the maturation process is exploited in tandem fluorescent protein timers (tFTs) to measure the dynamics of various cellular processes (Khmelinskii et al., 2012). A tFT is a fusion of two FPs that differ in maturation kinetics and spectral properties (i.e., color)-for example, a slower-maturing red fluorescent protein (redFP) and a fastermaturing green fluorescent protein (greenFP; Figure 1A). The greenFP is typically selected to be as rapidly maturing as possible and thus reports on protein localization and abundance. Because of the difference in maturation kinetics between the greenFP and redFP moieties, the color of a tFT (defined as the redFP/greenFP ratio of fluorescence intensities) changes over time ( Figure 1A). When a tFT is fused to a protein of interest, its color provides a measure of protein age that can be used to follow protein degradation, trafficking, and segregation during cell division (Khmelinskii et al., 2012).The range of protein ages that can be analyzed with a tFT, that is, its time range, is strongly influenced by the maturation kinetics of the slower-maturing FP in the pair (Khmelinskii et al., 2012). Therefore, in the tFTs described thus far (Khmelinskii et al., 2012;Khmelinskii and Knop, 2014;Dona et al., 2013), different slowermaturin...

Substrate Ubiquitination Controls the Unfolding Ability of the Proteasome

“…However, there are also reports that GFP can be difficult to unfold and degrade, perhaps because of its stable ␤-barrel structure (12,38,39), and in cells degradation of ubiquitin-GFP fusions are dependent on the action of the chaperone p97 (40,41). In particular, Saeki et al (12) reported that GFP resists degradation when tagged with K63-linked polyubiquitin chains, whereas other proteins were degraded efficiently.…”

Bicoid gradient formation mechanism and dynamics revealed by protein lifetime analysis

“…A further potential issue with tFT reporter measurements is incomplete degradation of the fluorescent timer (Khmelinskii et al, 2012(Khmelinskii et al, , 2016. If some fraction of free GFP protein survives the degradation of the tFT-Bcd protein, then the ratio measurements would be affected.…”

Mechanisms of up-regulation of Ubiquitin-Proteasome activity in the absence of NatA dependent N-terminal acetylation

“…These reporters consisted of an N-terminal ubiquitin followed by two variable residues (X and Z), a linker sequence (eK) and a tandem fluorescent protein timer (tFT). The tFT reports on protein stability independently of expression through the intensity ratio of the slow maturing mCherry and the fast maturing sfGFP fluorescent proteins, which increases as a function of protein half-life in steady state (Khmelinskii et al, 2012;Khmelinskii et al, 2016). In the course of that study we observed that reporters with a proline residue immediately following the ubiquitin moiety (Ubi-PZ-tFT reporters) exhibited increased turnover in strains lacking the N-terminal acetyltransferase NatA ( Fig 1A), whereas no destabilization was observed in NatB and NatC mutants (see Supplementary Figure S3 in (Kats et al, 2018)).…”

“…pdr5Δ ubr2Δ yeast cells expressing transcriptionally inactive Rpn4C477A mutants (Wang et al, 2004) C-terminally tagged with 10xHis-sfGFPcp8 (Khmelinskii et al, 2016)from a GPD promoter were grown in SC-His to 7x10 6 -8x10 6 cells/ml. Bortezomib was added to 50 µM and cultures were incubated for 1 h. 2.5x10 9 cells were harvested by centrifugation, washed with 20% (w/v) trichloroacetic acid, and stored at -80°C.…”