Protein stability and folding kinetics in the nucleus and endoplasmic reticulum of eucaryotic cells

Abstract

We measure the stability and folding relaxation rate of phosphoglycerate kinase (PGK) Förster resonance energy transfer (FRET) constructs localized in the nucleus or in the endoplasmic reticulum (ER) of eukaryotic cells. PGK has a more compact native state in the cellular compartments than in aqueous solution. Its native FRET signature is similar to that previously observed in a carbohydrate-crowding matrix, consistent with crowding being responsible for the compact native state of PGK in the cell. PGK folds through multiple states in vitro, but its folding kinetics is more two-state-like in the ER, so the folding mechanism can be modified by intracellular compartments. The nucleus increases PGK stability and folding rate over the cytoplasm and ER, even though the density of crowders in the nucleus is no greater than in the ER or cytoplasm. Nuclear folding kinetics (and to a lesser extent, thermodynamics) vary less from cell to cell than in the cytoplasm or ER, indicating a more homogeneous crowding and chemical environment in the nucleus.

Figure 1

Schematic diagram of the FReI setup. Living cells expressing fluorescent-labeled PGK are placed on coverslips on the microscope stage. Cells are illuminated using a blue LED (λ = 470 nm) and the resulting two-color fluorescence is split using dichroic mirrors and imaged simultaneously onto a CCD camera. Folding/unfolding dynamics are initiated by a shaped heating pulse from an infrared diode laser (λ = 2200 nm).

Figure 2

(A) Schematic energy landscape showing how protein populations shift upon a temperature jump. (B) Measured quantum yield trends (circles) of AcGFP1 and mCherry as a function of temperature, and linear fits (dotted lines). (C) Thermal denaturation of PGK (circles) represented in two different schemes, with and without linear baselines.

Figure 3

Fluorescence images showing subcellular localization of (A) FRET-PGK^∗, (B) FRET-PGK^∗-NLS, and (C) FRET-PGK^∗-ER. AcGFP1 has a quantum yield of 0.8, whereas mCherry has a quantum yield of 0.2, so the protein appears green at elevated temperature.

Figure 4

Representative experimental thermal melts (circles) of PGK in vivo in the cytoplasm, ER, and nucleus. A representative in vitro trace is shown for PGK-FRET-ER^∗. Fits to the cooperative model in the text are shown as lines.

Figure 5

Representative kinetic traces for PGK folding kinetics in the nucleus, ER, and cytoplasm (A) in vivo (open circles) and (B) in vitro (solid circles). The lines represent fits to the data using Eq. 10.

Figure 6

Plot of the melting temperature T_m versus relaxation time τ (at ∼T_m) for FRET-labeled PGK in various cellular organelles and in vitro. In vitro, different tagged PGKs have similar values of both T_m and τ. In vivo, the cytoplasm and the ER provide similar folding environments, whereas the nuclear environment stabilizes the protein and makes it fold faster relative to the cytoplasm and ER. The individual data points with 1 SD error bars are measurements of T_m and τ carried out for the same cell, while the dotted ellipses include additional cells for which only T_m or τ was measured. The widths of the ellipses are the spreads (from Tables 2 and 3) for the whole ensemble of cells measured.