TM5 (MATα pho3 pho5 trp1 leu2 ura3 his3) cells were selected as Yeast Saccharomyces cerevisiae. To be expressed a large amount of PDI, TM5 was harboring multicopy plasmid Yep13 containing the PDI1 gene. The yeast cells were grown at 24˚C for 18 h and harvested. For radioisotope labelling cells in Yeast, Saccharomyces cerevisiae X2180-1A (MAT α, gal 2, cup 1) strain and temperature sensitive mutant HMSF176 (MAT α, sec 18-1) strain were used.

To investigate the intracellular location of the PDI in yeast, yeast cell extracts were fractionated in sucrose density gradient as described by Ruohola et al. [9]

Yeast cells (100 OD₆₀₀ units) were then resuspended in 1 mL of the spheroplast medium (1.4 M sorbitol, 50 mM potassium phosphate, 56 mM β-mercaptoethanol, pH 7.5) containing zymolyase (1 mg/mL) and converted to spheroplasts for 1 h at 38˚C. After centrifuging these sedimented spheroplast mixtures in sorbitol cushion (1.7 M sorbitol, 20 mM sodium phosphate, pH 7.5), purified spheroplasts were resuspended in 4 mL of the lysis buffer (0.3 M mannitol, 10 mM Mops, pH 7.0) containing N, N’-diphenyl-p-phenylene diamine (0.1 mg/mL) and aprotinin (100 U/mL). The homogenization was also performed according to Ruohola et al. [9], and finally, 3 mL of homogenate in 60% (W/W) sucrose was obtained. Two milliliter of homogenate was placed at the bottom of an ultracentrifuge tube and overlaid with the following sucrose solutions: 1 mL of 55% sucrose, 1.5 mL each of 50%, 45%, 40% and 35% sucrose and 2 mL of 30% sucrose. The gradient was centrifuged at 4˚C for 12 h at 170,000 × g and fractionated in microtubes. A total of 23 factions, each about 500 μL, were collected in microtubes using a gradient tube fractionator, and then, the density of sucrose in each fraction was determined by refract meter.

As marker enzymes of the distribution, NADPH-cytochrome c reductase, Kex2 and α-mannosidase were used. Their assays were directly performed as described [10 - 12], the PDI assay was performed as described by Mizunaga et al. [7] after buffer of each fraction was replaced to 50 mM sodium phosphate buffer (pH 7.5) containing 50 mM EDTA.

Saccharomyces cerevisiae X2180-1A (MAT α, gal 2, cup 1) strain and temperature sensitive mutant HMSF176 (MAT α, sec 18-1) strain [13] grown at 24˚C were labeled with ³⁵S (Trans ³⁵S-label Metabolic Labeling Reagent, ICN) for 30 min at 24˚C or 37˚C and collected as described in reference [5]. After washing once with 10 mM sodium azide, radiolabeled cells were resuspended at 100OD₆₀₀ units/mL in 50 mM Tris-HCl buffer (pH7.5) containing l% SDS and 1 mM phenylmethylsulfonylfluoride and homogenized as described in reference [7]. Cell extracts were boiled for 5 min, and debris were removed by centrifugation for 5 min at 6000 × g. Two hundred microliter of respective supernatants were diluted with 800 μL of ice cold 1.25% TritonX-100 in 60 mM Tris-HCl buffer (containing 190 mM NaCl and 6 mM EDTA, pH7.4) and immunoprecipitated for 12 h at 4˚C by addition of 10 μL of anti-PDI antibody [7] followed by addition of 50 μL of 20% protein A-Sepharose suspension. Samples were rotated at room temperature for 2 h.

Immnunoprecipitates were washed twice with 1 mL RIPA buffer (0.5 M NaCl; 50 mM Tris-HCl [pH7.4]; 0.5% NP-40; 0.5% deoxycholate; 0.1% SDS), followed by two washes with PBS. At this stage, each sample was either divided for SDS-PAGE or for reimmunoprecipitation against anti-α-1,6 mannose antibody. For SDS-PAGE, samples were then resuspended in SDS sample buffer, boiled for 5 min and analyzed by SDS-PAGE. For the second immnunoprecipitation, anti-PDI antibody reactive proteins were eluted from the resin by adding 50 μL of 1% SDS in 50 mM Tris-HCl (pH 7.4) and boiled for 5 min. Eluted proteins were reprecipitated with the appropriate anti-α-1,6 mannose antibody and then also analyzed by SDS-PAGE as described above. The electrophoresed gel was treated with Amplify, dried and exposed to an X-ray film.

The result was shown in Figure 1. Activities of PDI and NADPH-cytochrome c reductase, known as an ER marker enzyme, co-migrated. Kex2, known as a Golgi marker enzyme, is eluted in the 5 - 6 fractions before the PDI’s peak fraction. α-Mannosidase, as a vacuole marker enzyme, is clearly separated from the activities of NADPH-cytochrome c reductase and PDI.

It has been also demonstrated that addition of α-1,6 linked mannose residues occurs especially in yeast early Golgi apparatus, like that of mammalian cells [13]. Anti-α-1,6 mannose antibody was used to determine whether yeast PDI obtains this Golgi-specific glycosylation and attempted to demonstrate recycling of PDI from the Golgi to the ER.

As shown in Figure 2, PDI extracted from X2180-1A cells radiolabeled at either 24˚C or 37˚C (lane 1 and 2) had acquired the α-1,6 mannose addition, as evidenced by the precipitation of α-1,6 mannose-containing material with anti-α-1,6 mannose antibody (lane 8 and 9).

Similarly, PDI extracted from secl8 mutant cells labeled at the permissive temperature (24˚C) (lane 3) also had acquired the α-1,6 mannose addition (lane 6). On the other hand, when sec18 cells were radiolabeled at the nonpermissive temperature (37˚C) (lane 4), PDI extracted from them showed no reactivity with anti-α-1,6 mannose antibody (lane 7). At the nonpermissive temperature, all vesicular traffic is

blocked in sec18 mutant, and newly synthesized proteins cannot leave the ER [14 , 15]. These results suggested that the luminal ER protein, PDI, had reached the early Golgi compartment and received α-1,6 mannose addition.