1. Yeast Lab 2 To do today: 1.Make lysates of WT, sec18 & sec61 strains grown at RT & 37°C. 2.Run lysates on 12% SDS- PAGE gel. 3.Score results from yeast growth assay (don’t look at predictions while doing this). 4.Take pictures of plates. 5.Transfer proteins from gel to nitrocellulose for Western blotting in Lab 3.

2. Yeast mating behavior All of our strains are MAT  and therefore produce  factor.

3. Features of  -factor Mature, secreted form of  -factor is a 13- amino acid polypeptide encoded by MF  1 gene (MW < 3.4 kDa) Precursor protein synthesized by the ribosome is much larger; maturation involves proteolytic processing in the Golgi & secretory vesicles –Prepro-  -factor (pp-  F) = entire polypeptide translated by ribosome (165 aa, 18.58 kDa MW) –Pro-  -factor = after removal of signal sequence, before proteolytic cleavage –pp-  F has three consensus sites for N- linked glycosylation (-Asn-X-Thr/Ser); the fully glycosylated form has a MW of 26 kDa Like most secreted proteins the  -factor signal sequence is removed in the ER (contrary to Julius et al. results) Ref: Julius, D. et al. (1984). Cell 36:309-318.

4. Review of Protein Glycosylation Two types: N-linked (modification of Asn residues) & O-linked (modification of Thr/Ser residues) —Core N-linked glycosylation & initial trimming occurs in ER; further modifications may occur in Golgi —O-linked occurs in Golgi Core N-linked glycosylation in the ER

5. Steps in the synthesis & maturation of  -factor Ref: Julius, D. et al. (1984). Cell 36:309-318.

6. OUR EXPERIMENT Observe the forms of pp  F that are present in WT, sec18, and sec61 cells grown at permissive & non- permissive temperatures by doing a Western blot using an anti-pp  F antibody (rabbit polyclonal). To Consider: Will we expect to see much intracellular pp  F in WT cells? What forms of pp  F do we expect to accumulate in the sec18 and sec61 mutants? Will these forms be present at both temperatures?

7. MF  1 gene product 1 MRFPSIFTAVLFAASSALAAPVNTTT EDETAQIPAEAVIGYLDLEGDFDVAV LPFSNSTNNGLLFINTTIASIAAKEEG VSLDKREAEAWHWLQLKPGQPMYK REAEAEAWHWLQLKPGQPMYKRE ADAEAWHWLQLKPGQPMYKREAD AEAWHWLQLKPGQPMY 165 1 2 3 4 red: glycosylation motifs (sugar attached to N) blue: mature  f sequence underline: peptide used for antibody synthesis

8. Preparing the yeast lysates Mechanical lysis in SDS sample buffer using glass beads & Fast Prep bead beater (QBiogene) Lysis done at 4°C to prevent proteolysis After lysis, boil samples 5 min to ensure denaturation (spin briefly before loading) Load gel as shown on p. 106 Run gel at 200V for 35 min (don’t want small proteins to run off)

9. Assembling the blot & doing the transfer black side of cassette