Fig. 1

Fig. 1

APEX-GBP Correctly Localizes to GFP-Tagged Subcellular Markers in BHK Cells

(A) Schematic of conventional APEX-tagging.

(B) Schematic of modular APEX tagging consisting of an APEX tag conjugated via linker domain to GFP-binding peptide.

(C) APEX-GBP expression alone is soluble and present only in the cytoplasm of transfected cells.

(D) APEX-GBP when co-transfected with GFP results in a similar distribution of DAB reaction product to APEX-GBP alone.

(E) GFP-Cav3^DGV is known to localize to lipid droplets; DAB reaction product was restricted to the surface of morphologically identifiable lipid droplets.

(F) Co-transfection of GFP-tK translocates the DAB reaction product to the PM. Left: unstained, central. Right: post-stained. Arrows indicate areas of enriched density at the PM. Post-staining demonstrates the quality of the preservation as this method improves the contrast of membranes but reduces the signal-to-noise ratio of the APEX-GBP. Post-staining reveals clathrin coated pits and caveolae at the surface of BHK cells.

(G) Rab5-GFP overexpression with APEX-GBP results in prominent endosomal staining that is tightly opposed to membrane: highlighted by arrows.

(H) Rab7-GFP overexpression with APEX-GBP results in significant DAB reaction at later-stage endosomes within transfected cells. Arrows indicate the accumulation of Rab7-GFP and APEX-GBP within endosomes.

(I) nls-GFP expression with APEX-GBP results in reaction product exclusively in the nucleus of transfected cells.

(J) GFP-vimentin was co-transfected with APEX-GBP and demonstrated significant electron density in perinuclear regions and in bundled fibril extensions into the cytoplasm.

Untransfected adjacent cells without reaction product, indicating the reaction product is specific. Cy, cytoplasm; Cav, Caveolae; CCP, clathrin-coated pit; M, mitochondria; PM, plasma membrane; LDs, lipid droplets; EE, early endosome; LEs, late endosomes; Nuc, nucleus; IFs, intermediate filaments. Scale bars represent 1 µm. The above localizations were confirmed by confocal microscopy; see Figure S1.