Yeast Lab 2
To do today:
1. Make lysates of WT, sec18 &
sec61 strains grown at RT &
37°C.
2. Run lysates on 12% SDSPAGE 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.
Yeast mating behavior
All of our
strains are
MAT a and
therefore
produce afactor.
Features of a-factor
• Mature, secreted form of a-factor is a 13amino acid polypeptide encoded by MFa1
gene (MW < 3.4 kDa)
• Precursor protein synthesized by the ribosome
is much larger; maturation involves
proteolytic processing in the Golgi &
secretory vesicles
– Prepro-a-factor (pp-aF) = entire
polypeptide translated by ribosome (165
aa, 18.58 kDa MW)
– Pro-a-factor = after removal of signal
sequence, before proteolytic cleavage
– pp-aF has three consensus sites for Nlinked glycosylation (-Asn-X-Thr/Ser); the
fully glycosylated form has a MW of 26
kDa
• Like most secreted proteins the a-factor
signal sequence is removed in the ER
(contrary to Julius et al. results)
Ref: Julius, D. et al. (1984). Cell 36:309-318.
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
Steps in the synthesis &
maturation of a-factor
Ref: Julius, D. et al. (1984). Cell 36:309-318.
OUR EXPERIMENT
Observe the forms of ppaF that are
present in WT, sec18, and sec61
cells grown at permissive & nonpermissive temperatures by doing a
Western blot using an anti-ppaF
antibody (rabbit polyclonal).
To Consider:
• Will we expect to see much intracellular ppaF
in WT cells?
• What forms of ppaF do we expect to
accumulate in the sec18 and sec61 mutants?
Will these forms be present at both
temperatures?
MFa1 gene product
1 MRFPSIFTAVLFAASSALAAPVNTTT
EDETAQIPAEAVIGYLDLEGDFDVAVLP
FSNSTNNGLLFINTTIASIAAKEEGVSL
1
DKREAEAWHWLQLKPGQPMYKREAEAEA
3
2
WHWLQLKPGQPMYKREADAEAWHWLQLK
4
PGQPMYKREADAEAWHWLQLKPGQPMY
165
red: glycosylation motifs (sugar attached to N)
blue: mature af sequence
underline: peptide used for antibody synthesis
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)
Assembling the blot &
doing the transfer
black side of
cassette