Lumenal endosomal and Golgi-retrieval determinants involved in pH-sensitive targeting of an early Golgi protein

Despite the potential importance of retrieval-based targeting, few Golgi ci sternae-localized proteins have been demonstrated to be targeted by retriev al, and the putative retrieval signals remain unknown. Golgi phosphoprotein of 130 kDa (GPP130) is a cis-Golgi protein that allows assay of retrieval- based targeting because it redistributes to endosomes upon treatment with a gents that disrupt lumenal pH, and it undergoes endosome-to-Golgi retrieval upon drug removal. Analysis of chimeric molecules containing domains from GPP130 and the plasma membrane protein dipeptidylpeptidase IV indicated tha t GPP130 targeting information is contained entirely within its lumenal dom ain. Dissection of the lumenal domain indicated that a predicted coiled-coi l stem domain adjacent to the transmembrane domain was both required and su fficient for pH-sensitive Golgi localization and endosome-to-Golgi retrieva l. Further dissection of this stem domain revealed two noncontiguous stretc hes that each conferred Golgi localization separated by a stretch that conf erred endosomal targeting. Importantly, in the absence of the endosomal det erminant the Golgi targeting of constructs containing either or both of the Golgi determinants became insensitive to pH disruption by monensin. Becaus e monensin blocks endosome-to-Golgi transport, the finding that the endosom al determinant confers monensin sensitivity suggests that the endosomal det erminant causes GPP130 to traffic to endosomes from which it is normally re trieved. Thus, our observations identify Golgi and endosomal targeting dete rminants within a lumenal predicted coiled-coil domain that appear to act c oordinately to mediate retrieval-based targeting of GPP130.