Detailed Analysis of the Interaction of Yeast COG Complex

Abstract

The Golgi apparatus is a central station for protein trafficking in eukaryotic cells. A widely accepted model of protein transport within the Golgi apparatus is cisternal maturation. Each cisterna has specific resident proteins, which are thought to be maintained by COPI-mediated transport. However, the mechanisms underlying specific sorting of these Golgi-resident proteins remain elusive. To obtain a clue to understand the selective sorting of vesicles between the Golgi cisterenae, we investigated the molecular arrangements of the conserved oligomeric Golgi (COG) subunits in yeast cells. Mutations in COG subunits cause defects in Golgi trafficking and glycosylation of proteins and are causative of Congenital Disorders of Glycosylation (CDG) in humans. Interactions among COG subunits in cytosolic and membrane fractions were investigated by co-immunoprecipitation. Cytosolic COG subunits existed as octamers, whereas membrane-associated COG subunits formed a variety of subcomplexes. Relocation of individual COG subunits to mitochondria resulted in recruitment of only a limited number of other COG subunits to mitochondria. These results indicate that COG proteins function in the forms of a variety of subcomplexes and suggest that the COG complex does not comprise stable tethering without other interactors.Key words: The Golgi apparatus, COG complex, yeast, membrane trafficking, multi-subunit tethering complex.

Keywords: COG complex; The Golgi apparatus; membrane trafficking; multi-subunit tethering complex; yeast.

Figure 1.

Interactions of COG subunits. Lysates from cells expressing Cog1-GFP (A), Cog3-GFP (B), Cog5-GFP (C) or Cog6-GFP (D) were separated into S100 (cytosol) and P100 (membrane) fractions by centrifugation at 100,000 × g. S100 and P100 fractions were immunoprecipitated (IP) with anti-GFP. IP and 10% immunodepleted/flow-through (FT) fractions were separated by SDS-PAGE and blotted with antibodies against Cog1–8. 10% input of solubilized proteins was run as a control. The bottom bar graph shows IP efficiency of upper blots. IP efficiency values were calculated by dividing IP by input.

Figure 2.

Growth of COG-mCherry-Fis1 expressing cells. (A) COG-Fis1 and COG-mCherry-Fis1 fusions. (B) COGs were expressed under GAL1 promoter by multi copy plasmids. Cells were grown on synthetic minus uracil plates containing 2% glucose or 2% galactose at 30˚C. (C) COG-mCherry-Fis1s were expressed under GAL1 promoter by plasmids integrated into chromosome. Cells were grown on YP plate containing 2% glucose or 2% galactose at 30˚C. (D) COG-Fis1s or COG-mCherry-Fis1s were expressed under GAL1 promoter by multi copy plasmids. Cells were grown on synthetic minus uracil plates containing 2% glucose or 2% galactose at 30˚C.

Figure 3.

COG-mCherry-Fis1 recruited other subunits to the mitochondria. COG-mCherry-Fis1s were expressed under GAL1 promoter by plasmids integrated into chromosome. Yeast cell lysates were immunoprecipitated (IP) with mCherry-Nanobody beads. IP and 5% immunodepleted/flow-through (FT) fractions were separated by SDS-PAGE and blotted with antibodies against mCherry, Cog1, 2, 4, 5, 6 and 8. 5% input of yeast total lysate was run as a control. The bottom bar graph shows IP efficiency of upper blots. IP efficiency values were calculated by dividing IP by input.

Figure 4.

Observation of Cog3- and Cog6-mCherry-Fis1 and SNARE proteins. (A) Cog3-mCherry-Fis1 and (B) Cog6-mCherry-Fis1 were expressed under GAL1 promoter by plasmids integrated into chromosome. GFP tagged SNARE proteins, Sed5, Gos1, Tlg1 and Tlg2 were constitutively expressed by low copy plasmids. Cells were grown in synthetic medium containing 1% raffinose overnight, then in synthetic medium containing 2% galactose for 3 h at 30˚C and observed by SCLIM. Maximum intensity projections were shown. Scale bar, 5 μm.