N-glycosylation enables high lateral mobility of GPI-anchored proteins at a molecular crowding threshold

The protein density in biological membranes can be extraordinarily high, but the impact of molecular crowding on the diffusion of membrane proteins has not been studied systematically in a natural system. The diversity of the membrane proteome of most cells may preclude systematic studies. African trypanosomes, however, feature a uniform surface coat that is dominated by a single type of variant surface glycoprotein (VSG). Here we study the density-dependence of the diffusion of different glycosylphosphatidylinositol-anchored VSG-types on living cells and in artificial membranes. Our results suggest that a specific molecular crowding threshold (MCT) limits diffusion and hence affects protein function. Obstacles in the form of heterologous proteins compromise the diffusion coefficient and the MCT. The trypanosome VSG-coat operates very close to its MCT. Importantly, our experiments show that N-linked glycans act as molecular insulators that reduce retarding intermolecular interactions allowing membrane proteins to function correctly even when densely packed.


Cumulative cell number (cells mL
We found that a final concentration of approx. 6 % (w/v) type A gelatin from porcine skin in TDB or PBS is well suited for immobilization of trypanosome BSF at 20 °C.
Trypanosomes were immobilized for different periods of time and subsequently recultivated to test the effect of the gelatin solution on cell viability. For this purpose, triplicates of 5 × 10 6 trypanosomes in 100 µL TDB were mixed with four volumes of 10 % gelatin solution and incubated at room temperature for either 0, 1, 2, 3, 4 or 7 hours. After incubation the cell-gelatin solution was heated to 37 °C. 100 µL of the cell-gelatin solution (1 × 10 6 cells) was suspended into a total volume of 10 mL HMI-9 medium. Subsequent cell growth was monitored for 72 hours using the standard cultivation procedure ( Supplementary Fig. 1).

Diffusion of the VSG is not impaired by gelatin
We analyzed the effect of 6 % (w/v) gelatin on the diffusion of VSG on supported membranes. For this purpose, VSGs were incorporated into supported membranes that had been spread on glass cover slips. Diffusion of VSGs was analyzed by FRAP in the presence and absence of 6 % (w/v) gelatin in PBS at 20 °C. Neither the diffusion coefficient nor the mobile fraction of VSG were reduced in the presence of solid gelatin (Supplementary Table 1).

Constructs and transgenic trypanosomes
For the generation of VSG121 and VSG117 N-glycosylation deficient cell lines VSG N-glycosylation signal peptides (N-X-S/T; X not P) were destroyed by site directed mutagenesis. VSG121 was mutated in position 434 of the mature protein to replace the endogenous threonine by alanine. VSG117 was mutated in position 422 of the mature protein to substitute threonine by alanine.

VSG overexpression
Overexpression of VSG was carried out using the tetracycline inducible cell line

Relative quantification of mfVSG in bilayers
Trypanosomes from an exponentially growing culture were washed three times with

FRAP of mfVSG on cells
Line-FRAP measurements of labeled trypanosomes were performed at a constant temperature of 20 °C. Ten pre-bleach and 100 post-bleach images were recorded at 2 fps. VSG diffusion half-life () and mobile fractions were determined using a single exponential function and data normalization according to Phair et al. 4 . The width (2x) of the lines bleached on the cell surface was determined by plotting the intensity profiles directly after bleaching. Diffusion coefficients were calculated by D = (x) 2 / (4 ). The diffusion coefficients and mobile fractions are means ± standard deviation.
The data were statistically validated with an unpaired and two tailed t-test (GraphPad Prism 5.0f).

FRAP of mfVSG on supported membranes
The diffusion coefficient and mobile fraction of the mfVSG on artificial membranes was determined by FRAP at a temperature of 20 °C. Typically 10 pre-bleach and 100 post-bleach frames were recorded at 2 sec per frame and 5 µm bleach spot radius.
Data analysis was performed according to Soumpasis et al. 5 .

Single-molecule Tracking
As the algorithm requires a starting guess for D in , which may also influence the resulting D out , a D in versus D out analysis was performed for 30 consecutive D in values in the range of 0.01 -4 (px 2 lag -1 ). D in was then chosen in a range where D out was independent of D in (Supplementary Fig. 5).