Julia Fueller | Search bioRxiv

bioRxiv2015

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

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 were constructed by combining different slower-maturing red fluorescent proteins (redFPs) with the same faster-maturing superfolder green fluorescent protein (sfGFP). Towards a comprehensive characterization of tFTs, we compare here tFTs composed of different faster-maturing 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 towards 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 design of new tFTs.\n\nAbbreviationstFT - tandem fluorescent protein timer\n\nFP - fluorescent protein\n\ngreenFP - green fluorescent protein\n\nredFP - red fluorescent protein

Cell Biology↗

bioRxiv2013

Journey to the Center of the Mitochondria Guided by the Tail Anchor of Protein Tyrosine Phosphatase 1B

The canonical protein tyrosine phosphatase PTP1B has traditionally been considered to exclusively reside on the endoplasmic reticulum (ER). Using confocal microscopy, we show that endogenous PTP1B actually exhibits a higher local concentration at the mitochondria in all mammalian cell lines that we tested. Fluorescently labeled chimeras containing full-length PTP1B or only its 35 amino acid tail anchor localized identically, demonstrating the complete dependence of PTP1Bs subcellular partitioning on its tail anchor. Correlative light and electron microscopy using GFP-driven photo-oxidation of DAB revealed that PTP1Bs tail anchor localizes it to the mitochondrial interior and to mitochondrial-associated membrane (MAM) sites along the ER. Heterologous expression of the tail anchor of PTP1B in the yeast S. cerevisiae surprisingly led to its exclusive localization to the ER/vacuole with no presence at the mitochondria. Studies with various yeast mutants of conserved membrane insertion pathways revealed a role for the GET/TRC40 pathway in ER insertion, but also emphasized the likely dominant role of spontaneous insertion. Further studies of modified tail isoforms in both yeast and mammalian cells revealed a remarkable sensitivity of subcellular partitioning to slight changes in transmembrane domain (TMD) length, C-terminal charge, and hydropathy. For example, addition of a single positive charge to the tail anchor was sufficient to completely shift the tail anchor to the mitochondria in mammalian cells and to largely shift it there in yeast cells, and a point mutation that increased TMD hydropathy was sufficient to localize the tail anchor exclusively to the ER in mammalian cells. Striking differences in the subcellular partitioning of a given tail anchor isoform in mammalian versus yeast cells most likely point to fundamental differences in the lipid composition of specific organelles (e.g. affecting membrane charge or thickness) in higher versus lower eukaryotes. Fluorescence lifetime imaging microscopy (FLIM) detection of the Forster Resonance Energy Transfer (FRET)-based interaction of the catalytic domain of PTP1B with the epidermal growth factor receptor (EGFR/ErbB1) at the mitochondria revealed a strong interaction on the cytosolic face of the outer mitochondrial membrane (OMM), suggesting the presence of a significant pool of PTP1B there and a novel role for PTP1B in the regulation of mitochondrial ErbB1 activity. In summary, in addition to its well-established general localization along the ER, our results reveal that PTP1B specifically accumulates at MAM sites along the ER and localizes as well to the OMM and mitochondrial matrix. Further elucidation of PTP1Bs roles in these different locations (including the identification of its targets) will likely be critical for understanding its complex regulation of general cellular responses, cell proliferation, and diseased states.

Cell Biology↗