The COG and COPI complexes interact to control the abundance of GEARs, a subset of Golgi integral membrane proteins

Abstract

The conserved oligomeric Golgi (COG) complex is a soluble hetero-octamer associated with the cytoplasmic surface of the Golgi. Mammalian somatic cell mutants lacking the Cog1 (ldlB) or Cog2 (ldlC) subunits exhibit pleiotropic defects in Golgi-associated glycoprotein and glycolipid processing that suggest COG is involved in the localization, transport, and/or function of multiple Golgi processing proteins. We have identified a set of COG-sensitive, integral membrane Golgi proteins called GEARs (mannosidase II, GOS-28, GS15, GPP130, CASP, giantin, and golgin-84) whose abundances were reduced in the mutant cells and, in some cases, increased in COG-overexpressing cells. In the mutants, some GEARs were abnormally localized in the endoplasmic reticulum and were degraded by proteasomes. The distributions of the GEARs were altered by small interfering RNA depletion of epsilon-COP in wild-type cells under conditions in which COG-insensitive proteins were unaffected. Furthermore, synthetic phenotypes arose in mutants deficient in both epsilon-COP and either Cog1 or Cog2. COG and COPI may work in concert to ensure the proper retention or retrieval of a subset of proteins in the Golgi, and COG helps prevent the endoplasmic reticulum accumulation and degradation of some GEARs.

Figure 1.

Immunoblotting and immunofluorescence localization of Golgi-associated proteins in wild-type CHO, mutant ldlB and ldlC, and transfected ldlB[COG1] and ldlC[COG2] cells. (A) Total cell lysates (20 μg of protein) were prepared from parental wild-type CHO, ldlB, ldlB[COG1] (ldlB cells transfected with COG1 cDNA, Cog1 overexpressed), ldlC and ldlC[COG2] (ldlC cells transfected with COG2 cDNA, Cog2 overexpressed) cells. Lysates were subjected to SDS-PAGE and immunoblotting by using antibodies to the indicated Golgi-associated proteins. Solid and open arrowheads indicated a 115-kDa band of GPP130 and two isoforms (42 and 35 kDa) of syntaxin-5, respectively. (B) The indicated cells (left) were grown at 37°C (except those stained with anti-CASP antibody, which were grown at 34°C), fixed with paraformaldehyde, and stained with antibodies to the indicated proteins. To permit comparison of signal intensities among the images in the same column, the images were collected with a fixed signal gain by confocal microscopy. Bar, 10 μm.

Figure 2.

Stability and intracellular distributions of GOS-28 in wild-type CHO, ldlB, and ldlC cells. (A) Cells were metabolically labeled with [³⁵S]methionine/cysteine for 30 min (pulse), and then either chased for the indicated times in medium B containing unlabeled methionine and cysteine (top) or incubated for 24 h with the same medium plus 25 μM of the proteasome inhibitors MG132 or lactacystin (bottom). Cell lysates were subjected to immunoprecipitation with an anti-GOS-28 antibody. The immunoprecipitates were fractionated by SDS-PAGE, and the bands were visualized by autoradiography. (B) Cells were fixed and stained with antibodies to GOS-28 (red) and the ER marker ERp72 (green). To visualize the distribution of GOS-28 by using confocal microscopy, the images from ldlB and ldlC cells (weaker signals) were collected with higher signal gains than those from CHO cells. Note that because of the location of the section, some of the confocal images of the mutants do not show the perinuclear Golgi ribbon staining that is present (e.g., see Figure 1B). Bar, 10 μm.

Figure 3.

Immunofluorescence localization of GOS-28 and GM130 in CHO, ldlB, and ldlC cells treated without and with lactacystin. (A) Cells were incubated for 8 h without (none) or with (+Lact) 25 μM lactacystin, fixed, and stained with antibodies to GOS-28 (red) and GM130 (green). To visualize more clearly the distribution of GOS-28 by using confocal microscopy, the images from ldlB and ldlC cells (weaker signals) were collected with higher signal gains than those from CHO cells. However, a fixed signal gain was used to collect images from any given cell type when comparing the effects of incubation without or with lactacystin. (B) The ldlB cells were preincubated with cycloheximide (+CHX, 140 μg/ml) for 1 h and then further incubated for 8 h without or with (+Lact) 25 μM lactacystin in the presence of the cycloheximide. Bar, 10 μm.

Figure 7.

Effects of siRNA depletion of ε-COP in ldlB and ldlC cells on the immunofluorescence of GOS-28, GM130, and ε-COP. ldlB and ldlC mutants were transfected without (control) or with (+siRNA) ε-COP–specific siRNA. Cells were incubated at 34°C for 48 h and then fixed and stained with antibodies to either GOS-28 (red) or GM130 (red) and ε-COP (green). To facilitate visualization of the distribution of GOS-28 by using confocal microscopy, the images from ldlB and ldlC cells (weaker signals) were collected with relatively high signal gains. Arrowheads indicate cells in which there was a loss or reduction the signal intensity for ε-COP. Bar, 10 μm.

Figure 4.

Steady-state levels of GOS-28, GS15, the subunits of COG and GM130 in untransfected and COG subunit-overexpressing CHO and ldlB cells. Total cell lysates from the CHO, ldlB, and ldlB[COG1] cells, as well as transfected CHO cells that overexpress exogenous Cog1, Cog2, or HA-tagged Cog7 (CHO[COG1], CHO[COG2] and CHO[HA-COG7]) were subjected to SDS-PAGE and immunoblotting. Open arrowhead indicates the HA-tagged Cog7 protein whose electrophoretic mobility is slightly less than that of the endogenous Cog7.

Figure 5.

Immunoblotting and immunofluorescence localization of the α-, β-, and ε-COP subunits of COPI. (A) Lysates from the indicated cells were analyzed by immunoblotting with antibodies to α-, β-, or ε-COP. (B) The indicated cells were fixed and stained with an anti-β-COP antibody, and images were collected by confocal microscopy as described in Figure 1. Bar, 10 μm.

Figure 6.

Effects of siRNA depletion of ε-COP in wild-type CHO cells on the immunofluorescence of Cog1, GOS-28, GM130, and β-COP. Wild-type CHO cells were transiently transfected without (control) or with (+siRNA) a 21-base pair siRNA duplex specific for hamster ε-COP. After a 46.5-h incubation at 34°C, the cells were further incubated for 90 min at either 34°C (where the loss of ε-COP had no apparent phenotype) or 39.5°C and then fixed and stained with antibodies to either ε-COP or β-COP (green) together with antibodies (red) to either Cog1, GM130, or GOS-28. Arrowheads indicate the cells in which there was a loss or major reduction in the signal intensity for either ε-COP (A and B) or GOS-28 (C), which indicated effective knockdown of ε-COP expression. Bars, 10 μm.