Both post-Golgi and intra-Golgi cycling affect the distribution of the Golgi phosphoprotein GPP130

Golgi phosphoprotein, GPP130, a cis Golgi protein, is representative of proteins cycling between the Golgi apparatus and endosomes in a pH-sensitive manner. The present qualitative data are insufficient to distinguish the relative contributions of Golgi and endosomal processes in regulating the cycling of such proteins. We have taken a quantitative approach to analyze GPP130 distribution in response to pH perturbation. We have used Shiga-like toxin B fragment, a protein that traffics from the cell surface and Golgi apparatus by the late endosomal bypass pathway, as a probe to highlight one aspect of GPP130 cycling and similarly the trafficking of tsO45-green fluorescent protein (GFP) between the Golgi apparatus and the plasma membrane to treat that aspect of GPP130 cycling in isolation. Overall, we conclude from quantitative analysis and simulations that treatment of HeLa cells with the pH perturbant, monensin, affects GPP130 cycling at several stages with effects on (i) intra-Golgi cycling, (ii) trans Golgi to endosome transport and (iii) endosome to Golgi transport. Our analysis indicates that the effect is greatest at the trans Golgi, the most acidic portion of the Golgi apparatus. In sum, multiple, regulated steps affect the trafficking of GPP130.