pH of TGN and recycling endosomes of H⁺/K⁺-ATPase-transfected HEK-293 cells: implications for pH regulation in the secretory pathway

Abstract

The influences of the gastric H+/K+ pump on organelle pH during trafficking to and from the plasma membrane were investigated using HEK-293 cells stably expressing the alpha- and beta-subunits of human H+/K+-ATPase (H+/K+-alpha,beta cells). The pH values of trans-Golgi network (pHTGN) and recycling endosomes (pHRE) were measured by transfecting H+/K+-alpha,beta cells with the pH-sensitive GFP pHluorin fused to targeting sequences of either TGN38 or synaptobrevin, respectively. Immunofluorescence showed that H+/K+-ATPase was present in the plasma membrane, TGN, and RE. The pHTGN was similar in both H+/K+-alpha,beta cells (pHTGN 6.36) and vector-transfected ("mock") cells (pHTGN 6.34); pHRE was also similar in H+/K+-alpha,beta (pHRE 6.40) and mock cells (pHRE 6.37). SCH28080 (inhibits H+/K+-ATPase) caused TGN to alkalinize by 0.12 pH units; subsequent addition of bafilomycin (inhibits H+ v-ATPase) caused TGN to alkalinize from pH 6.4 up to a new steady-state pHTGN of 7.0-7.5, close to pHcytosol. Similar results were observed in RE. Thus H+/K+-ATPases that trafficked to the plasma membrane were active but had small effects to acidify the TGN and RE compared with H+ v-ATPase. Mathematical modeling predicted a large number of H+ v-ATPases (8000) active in the TGN to balance a large, passive H+ leak (with PH approximately 10-3 cm/s) via unidentified pathways out of the TGN. We propose that in the presence of this effective, though inefficient, buffer system in the Golgi and TGN, H+/K+-ATPases (estimated to be approximately 4000 active in the TGN) and other transporters have little effect on luminal pH as they traffic to the plasma membrane.

A Fluorescent Probe of Polyamine Transport Accumulates into Intracellular Acidic Vesicles via a Two-step Mechanism

“…A preferential accumulation of Spd-C 2 -BODIPY in the TGN may reflect a targeted expression of the putative vesicular polyamine transporter. Alternatively, such compartmentalization might result from sorting and recycling of that carrier by the TGN from vesicles arising from the late endocytic compartment (42) or could be a mere consequence of the ⌬pH dependence of vesicular polyamine transport, because the intracisternal pH of the TGN is ϳ5.9 (as compared with a luminal pH of 6.2-6.6 in the cis to medial regions of the Golgi) (43).…”

The ER-Golgi intermediate compartment (ERGIC): in search of its identity and function

“…3). Although its exact pH is unknown, it is unlikely to be lower than that of the trans-Golgi network (pH 6.4) (Machen et al, 2003). Even if only slightly lower than in the ER, the reduced pH in the ERGIC may act as a general trigger for the dissociation of cargo-receptor complexes in combination with other factors.…”

Renal Vacuolar H+-ATPase

“…Both immunocytochemical and functional studies have demonstrated the presence of proton pumps on endosomal membranes and parts of the Golgi/TGN (205,238,328,345,346,355,365,442) as well as on lysosomal membranes (366). Some of the effects of increasing luminal pH in intracellular compartments are well understood.…”