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 a cis-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.

Figure 1

Schematic representation of GPP130/DPPIV chimeras. GPP130 sequences are represented by open boxes, DPPIV sequences are shaded, and the HA epitope is filled. Names of constructs are based on GPP130 amino acid numbering. Note that all constructs have cytoplasmic (cyto) and transmembrane (TM) domains. The localization of each chimera after no treatment (Ø), monensin treatment (mon), and monensin washout (w/o) is indicated to the right as either Golgi (G), endosome (E), plasma membrane (PM), or unchanged (∗). At the top is a plot of the predicted GPP130 coiled-coil domains derived by the COILS program (Lupas et al., 1991) with a window size of 28.

Figure 2

GPP130 lumenal domain is required and sufficient for Golgi targeting. CHO cells transfected with constructs for full-length GPP130 (A), full-length DPPIV (B), chimera 1–38 containing GPP130 cytoplasmic and transmembrane domains (C), and chimera 40–696 containing the GPP130 lumenal domain (D) were treated with cycloheximide, fixed, and stained with the use of antibodies against GPP130 (A and D) or DPPIV (B and C). Untransfected CHO cells did not stain with either antibody. Bar, 10 μm.

Figure 3

Predicted coiled-coil stem domain (aa 38–248) within the GPP130 lumen is necessary and sufficient for Golgi localization. HeLa cells transfected with chimera 295–696 containing the acidic region (A and B); chimera 38–248 containing the coil domain (C and D); and Δ40–247, a version of GPP130 lacking the coil domain (E and F), were treated with cycloheximide for 4 h then fixed and double stained with the use of anti-HA (A, C, and E) and anti-giantin (Gtn; B, D, and F). Bar, 10 μm.

Figure 4

Predicted GPP130 coiled-coil stem mediates reversible redistribution to endosomes. HeLa cells transfected with the coil chimera 38–248 were incubated in 10 μM monensin in normal medium for 1 h (A and B) or 10 μM monensin for 1 h followed by washing and incubation in normal medium for 3 h (C and D). All incubations contained 100 μg/ml cycloheximide to prevent new protein synthesis. The cells were then fixed and double stained with anti-HA (A and C) and anti-giantin (B and D). Bar, 10 μm.

Figure 5

Separation of endosomal and Golgi-targeting determinants. HeLa cells transfected with chimera 38–107 (A and B), chimera 89–175 (C and D), or chimera 176–248 (E and F) were cycloheximide treated, fixed, and double stained with the use of anti-HA (A, C, and E) and anti-giantin (B, D, and F). Bar, 10 μm.

Figure 6

Internalized FITC-dextran partially colocalizes with the endosomal determinant-containing chimera. COS-7 cells transfected with chimera 89–175 were incubated for 30 min with 1 mg/ml fixable FITC-dextran. After fixation the anti-HA staining pattern (A) was compared with the FITC pattern (B). Arrowheads mark a subset of the many puncta that were costained. These cells were not cycloheximide treated. Bar, 10 μm.

Figure 7

Golgi-localized chimeras do not stably interact with endogenous GPP130. Nonionic detergent lysates of cells transfected with either the 89–294 chimera (top panels) or the 176–248 chimera (bottom panels) were incubated with immobilized anti-GPP130. Bound and unbound material was collected and analyzed by immunoblotting to detect GPP130 and the chimeric proteins as indicated. Note that there was no detectable chimeric protein associated with immunoprecipitated GPP130, even though GPP130 was essentially depleted from the lysates.

Figure 8

GPP130 endosomal determinant is required for pH-sensitive Golgi targeting. HeLa cells transfected with chimera 38–107 containing a Golgi determinant (A and B); chimera 176–248 containing a Golgi determinant (C and D); or Δ89–175, a version of GPP130 lacking the endosomal determinant (E and F), were treated with 10 μM monensin for 1 h in the presence of cycloheximide. They were then fixed and double stained with the use of anti-HA (A, C, and E) and anti-giantin (B, D, and F). Unlike wild type, chimeras with either of the isolated Golgi determinants remained Golgi localized after monensin treatment. This was also the case for GPP130 lacking the endosomal determinant. Bar, 10 μm.

Figure 9

Endosomal determinant confers pH sensitivity to the isolated GPP130 Golgi determinants. HeLa cells transfected with chimera 38–175 (A–C) or chimera 89–248 (D–F) were incubated in normal medium 4 h (A and D), incubated in 10 μM monensin in normal medium for 1 h (B and E), or incubated in 10 μM monensin for 1 h followed by washing and incubation in normal medium for 3 h (C and F). All incubations contained 100 μg/ml cycloheximide to prevent new protein synthesis. The cells were then fixed and double stained. Anti-HA staining is shown. The presence of the endosomal determinant restored the monensin-induced reversible redistribution to endosomes. Bar, 10 μm.