Quantitative analysis of endocytosis with cytoplasmic pHluorin chimeras

Abstract

The pH-sensitive green fluorescent protein (GFP) variant pHluorin is typically fused to the extracellular domain of transmembrane proteins to monitor endocytosis. Here, we have turned pHluorin inside-out, and show that cytoplasmic fusions of pHluorin are effective quantitative reporters for endocytosis and multivesicular body (MVB) sorting. In yeast in particular, fusion of GFP and its variants on the extracellular side of transmembrane proteins can result in perturbed trafficking. In contrast, cytoplasmic fusions are well tolerated, allowing for the quantitative assessment of trafficking of virtually any transmembrane protein. Quenching of degradation-resistant pHluorin in the acidic vacuole permits quantification of extravacuolar cargo proteins at steady-state levels and is compatible with kinetic analysis of endocytosis in live cells.

Figure 1. Transit of cargo bearing a cytoplasmic tag through the endocytic pathway

During endocytosis, transmembrane proteins retain their topology with respect to the cytoplasm and the extracellular space or vesicle lumen. However, membrane-associated proteins destined for degradation in the vacuole are often packaged into internal vesicles within the multivesicular body (MVB) via the ESCRT machinery. Upon fusion of MVBs with the vacuole, the internal vesicles are exposed to vacuolar hydrolases, and their contents are degraded. Although a cytoplasmic GFP tag (left) can be delivered to the vacuole in such a manner, it retains detectable fluorescence within the vacuole (denoted as a green tag) because of resistance to degradation by vacuolar proteases and changes in pH. In contrast, a cytoplasmic pHluorin tag (right), which is similarly resistant to degradation but is sensitive to changes in pH, loses its fluorescence (denoted as a black tag) upon incorporation into internal MVB vesicles and delivery to the acidic vacuole.

Figure 2. Quenching of a pHluorin-tagged endocytic cargo protein in the vacuole lumen

Wild-type (WT), ΔΔΔΔ + Ent1 and ΔΔΔΔ + ENTH1 cells tagged with C-terminal GFP (left panels) or pHluorin (right panels) at the STE3 locus. For each fluorescent tag, any adjustment to the brightness or contrast of images was identical for all strains in order to accurately reflect differences in fluorescence intensity. Furthermore, all images were captured under identical exposure conditions. Corresponding differential interference contrast (DIC) images are presented for all panels.

Figure 3. pHluorin used for quantification of steady-state levels of extravacuolar cargo by fluorescence microscopy

Fluorescence intensity (in arbitrary units, a.u.) of WT, ΔΔΔΔ + Ent1 and ΔΔΔΔ + ENTH1 cells expressing Ste3-GFP (A) or Ste3-pHluorin (B). Values are presented as mean ± s.e.m., with the number of measured cells indicated in parentheses (One-way ANOVA with Tukey’s Multiple Comparison post-hoc test, ***p<0.001). (C) Western immunoblot of cell extracts from WT, ΔΔΔΔ + Ent1 and ΔΔΔΔ + ENTH1 strains tagged at the endogenous STE3 locus with either GFP or pHluorin and probed with anti-Ste3 or anti-GAPDH antibodies as indicated.

Figure 4. pHluorin used for kinetic analysis of endocytosis

(A) Cells tagged with Mup1-GFP or Mup1-pHluorin were imaged in the absence (− Met) or presence (+ Met) of methionine at the indicated times. For each series, maximum and minimum intensities were normalized to the same level for each image in order to reflect changes in the relative Mup1p fluorescence over time. (B) Total cellular fluorescence of Mup1-GFP or Mup1-pHluorin was measured at 5 min intervals from cells growing in the absence (− Met, for Mup1-GFP and for Mup1-pHluorin) or presence (+ Met, for Mup1-GFP and for Mup1-pHluorin) of methionine. Fluorescence intensity was normalized to the first time point of the series, and values are presented as mean ± s.d. (n=6).

Figure 5. Detection of fluorescently-tagged Ste3p in protease-deficient cells

(A) WT and protease-deficient (pep4Δ, prb1Δ, prc1Δ) cells expressing GFP-or pHluorin-tagged Ste3p were imaged by fluorescence microscopy, with maximum and minimum intensity adjustments applied equally to all samples to demonstrate the relative fluorescence intensity in each strain. (B) Total cellular fluorescence was measured for individual cells from the strains shown in panel A. Values are presented as mean ± s.e.m., with the total number of cells measured per condition indicated in parentheses. (C) Western immunoblot of cell extracts from WT and protease-deficient strains tagged with either GFP or pHluorin at the endogenous STE3 and probed with anti-Ste3 or anti-GAPDH antibodies as indicated.

Figure 6. Manipulation of vacuolar pH or delivery into multivesicular bodies affects quenching of luminal pHluorin

(A) pHluorin-tagged cells treated with vehicle (DMSO) or Bafilomycin-A1 (Baf-A1). Arrows indicate bright vacuoles. (B) GFP- and pHluorin-tagged cells expressing dominant-negative Vps4^E233Q. Arrows indicate fluorescence at the vacuolar limiting membrane. Corresponding differential interference contrast (DIC) images are presented for all panels.

Figure 7. Acidificatioin of MVB vesicles detected with Ste3-pHluorin

Cells endogenously tagged with Ste3-GFP or Ste3-pHluorin and expressing Ist1-mCherry were treated for 10 min in the presence of vehicle (DMSO) or Bafilomycin-A1 (Baf-A1) prior to imaging by fluorescence microscopy.