Lumenal Endosomal and Golgi-Retrieval Determinants Involved in pH-sensitive Targeting of an Early Golgi Protein

Abstract

Despite the potential importance of retrieval-based targeting, few Golgi cisternae-localized proteins have been demonstrated to be targeted by retrieval, and the putative retrieval signals remain unknown. Golgi phosphoprotein of 130 kDa (GPP130) is acis-Golgi protein that allows assay of retrieval-based targeting because it redistributes to endosomes upon treatment with agents 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 that GPP130 targeting information is contained entirely within its lumenal domain. Dissection of the lumenal domain indicated that a predicted coiled-coil stem domain adjacent to the transmembrane domain was both required and sufficient for pH-sensitive Golgi localization and endosome-to-Golgi retrieval. Further dissection of this stem domain revealed two noncontiguous stretches that each conferred Golgi localization separated by a stretch that conferred endosomal targeting. Importantly, in the absence of the endosomal determinant the Golgi targeting of constructs containing either or both of the Golgi determinants became insensitive to pH disruption by monensin. Because monensin blocks endosome-to-Golgi transport, the finding that the endosomal determinant confers monensin sensitivity suggests that the endosomal determinant causes GPP130 to traffic to endosomes from which it is normally retrieved. Thus, our observations identify Golgi and endosomal targeting determinants within a lumenal predicted coiled-coil domain that appear to act coordinately to mediate retrieval-based targeting of GPP130.

Mouse RIC-3, an Endoplasmic Reticulum Chaperone, Promotes Assembly of the α7 Acetylcholine Receptor through a Cytoplasmic Coiled-Coil Domain

“…In contrast, mRIC-3 did not colocalize well with the fluorescently labeled Golgi marker, WGA (Virtanen et al, 1980;Li et al, 1995) (Fig. 2 AgAi) or with GPP130 (Linstedt et al, 1997;Bachert et al, 2001;Puri et al, 2002) a transmembrane protein specific for the cis-Golgi (Fig. 2 Aj-Al ).…”

TNO1 Is Involved in Salt Tolerance and Vacuolar Trafficking in Arabidopsis

“…A connection between salt sensitivity and vacuolar trafficking has been well established. In yeast and mammalian cells, post-Golgi organelles involved in vacuolar trafficking were shown to be targets for salt stress, resulting in inefficient vacuolar trafficking (Bachert et al, 2001;Pardo et al, 2006;Hernández et al, 2009). In Arabidopsis, the endocytic pathway to the vacuole has also been shown to be important for salt tolerance (Leshem et al, 2006(Leshem et al, , 2007.…”

Mitogen-activated Protein Kinase Kinase 1-dependent Golgi Unlinking Occurs in G2Phase and Promotes the G2/M Cell Cycle Transition

“…Primary antibodies used were as follows: anti-ERK2 C16 (Santa Cruz Biotechnology, Santa Cruz, CA), anti-GPP130 (Bachert et al, 2001), anti-p115 (Puthenveedu and Linstedt, 2001a), anti-GRASP65 (Wang et al, 2003), diphospho-specific anti-ERK1/2 (Santa Cruz Biotechnology), anti-hemagglutinin (HA) (Bachert et al, 2001), anti-Myc 9e10 (Jesch et al, 2001a), and anti-giantin (Linstedt and Hauri, 1993). Secondary antibodies were affinity purified and fluorescein isothiocyanate or rhodamine isothiocyanate conjugated (Invitrogen, Carlsbad, CA).…”