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Gabriel Gherman Bendavit
Department of Biology
Concordia University
In the secretion pathway, TRAPP I located in the Cis
Golgi) interacts with COPII coated vesicles in the ER-cis
Golgi pathway. TRAPP II is located in the trans-Golgi and
tether COP I coated vesicles from the early endosome1.
Vesicle coated proteins (COP I, COPII and Clathrin) act
in vesicle specificity.
TRAPP relevance and role in the secretion pathway
Introduction
TRAPP structure and role in the secretion pathway
Screening Methods Results
Role of TRS23 C terminal from a GEF Ypt/Rab
GTPase study
Workflow of the experiment
Yeast transformation
Mutated TRS23 With High Copy Plasmid Screen status
Acknowledgments
Mutant strain
Supression of mutant Phenotype
Conclusion
CIN 5 selection
The trs23Δ99C haploid S.Cerevisiae (MSY441b
strain type α) was transformed by a high copy
plasmid (Yep 24) with fragments of a high copy
plasmid yeast genomic library (6000 genes) which
generated 36000 colonies . The thermic sensitivity
phenotype (slow growth at 160C) was suppressed in
37 colonies. A positive control was used with another
high copy plasmid (prs 426) with TRS 23 gene on it.
Also, a negative control (prs 426 without yeast
genomic fragment) was used.
Slow growth
at 160C (severe)
After yeast transformation, colonies were replica
plated in three temperatures, permissive (300C),
hot (380C) and cold (160C). Using suppression
of thermic sensitivity phenotype, 15 colonies
were selected to be sequenced.
The role of CIN 5 as the agent suppressor of the mutant
phenotype is so far not conclusive. Its selection was
based in the relationship between TRS 23 and
Chromosome instability described by Ben-Aroya 2008.
In order to follow up on the study, CIN 5 needs to be
isolated and transformed into the trs23Δ99C mutant
yeast.
The activated form of Ypt/Rab is
when (GTP) is bound to Ypt/Rab
and its deactivated form is when
(GDP) is bound. The elements
with GEF activity work by
increasing the phosphorylation
speed of Ypt/Rab GDP and
phosphorylation to its activated
form Ypt/Rab GTP. Both
TRAPPI/II are GEF for Ypt/Rab
GTP ase.
Dr. Michael Sacher and all the great scientists
from his laboratory, especially to Stephanie
Brunet.
Confirmation of CIN5 suppression was done by
digesting this gene from Yep 24 with Eco R1 and
Xho1. The insert was then ligated into the vector
prs 426. After bacteria and yeast transformation,
the colonies will be evaluated for mutant phenotype
suppression. Unfortunately no growth after bacteria
transformation was obtained yet.
The 37 colonies with mutant phenotype
suppressed , selected from the 36,000
transformed yeast colonies, were transformed
into E coli (to increase number of copies). The
plasmids were then transformed back to yeast
(MSY441b) to reevaluate suppression of the
phenotype. A selected gene was used in a new
round of the screening ( digested from Yep24
and ligated in a new plasmid (prs 426 without
TRS23).
Slow growth
at 380C (mild)
After PCR amplification, 15 colonies with
mutant suppressed phenotype were sent to
be sequenced in three different batches.
The six samples that came back as empty
vectors will be transformed back into yeast.
One failed to be sequenced and an other
showed no ORFs. The seven samples with
yeast fragments summed a total of 21
genes. From those, CIN5 from
Chromosome XV was selected.
Membrane trafficking between enclosed cellular compartments
use vesicle-mediated transport in order to deliver secretory
proteins. One of the components involved in cargo selectivity
and proper fusion of vesicles with its specific target membrane
are multi-subunit complexes called tethering factors. The
transport protein particle (TRAPP) complex is a tethering factor
conserved from yeast to man that exists in two types TRAPP I,
in the cis Golgi and TRAPP II, in the trans Golgi . TRAPP I has
seven subunits and TRAPPII has three extra subunits, they
tether COPII and COPI coated protein respectively.2 In yeast
both TRAPPI/II are guanine nucleotide exchange factors (GEF)
for Ypt1/Rab GTPase which activates it. Ypt1/Rab is
responsible for vesicle formation, motility, tethering and fusion
of vesicles1. The TRAPP I subunit TRS 23 is a centrally
localized essential subunit which is a GEF for YPT1/ RAB
GTPase. A study with a C terminal mutated TRS 23 raised
some questions regarding this subunit interactions with the
TRAPPI complex.
Full understanding of TRS 23 function, such as GEF activity for
YPT1/ RAB GTPase, is fundamental for the study of the
mechanisms involved with the secretion pathway. For this
reason a high copy screening of the trs23Δ99C was done.
In a previous study3 a triple mutation on C terminal of TRS
23 was found to be deleterious for the cell with a reduction
of GEF activity in vitro.
TRAPP I complex subunit TRS 23
is centrally localized and
together with Bet 5 have a
binding site, showed by crystal
structure, for Ypt/Rab.
However, when a complete C
terminal deletion was done at
Dr Sacher’s laboratory the cell
was still viable. Also, a
destabilization in the TRAPP I
complex was revealed.
This mutation presented
thermic sensitivity and this
mutant phenotype was used
for the screen of Trs23Δ99C .
After bacteria transformation by heat shocking or
electroporation, the plasmids were extracted and
transformed back to yeast for confirmation of
mutant phenotype suppression.
CIN 5 (from the chromosome XV) encodes for
chromosome instability(CIN) and also salt and
antibiotic resistance.
The CIN 5 selection was based in the association of
TRS 23 with CIN. In one study TRS 23 was
identified by a screening that used a temperature-
sensitive allele for each essential gene in S.
cerevisiae. The mutant TRS 23 showed CIN4.
CIN 5 Ligation
References
Spondyloepiphyseal dysplasia tarda (SEDT) is one
example of TRAPP importance for human health. This
musculoskeletal disorder is associated with TRAPP
subunits of its mammalian homolog TRAPPC2.
The SNARE complex is
responsible for vesicle docking by
interaction of two of its subunits
t SNARE (in the membrane) and v
SNARE ( in the vesicle ). TRAPP I
subunit Bet3 binds to the COPII
subunit Sec 23 allowing t SNARE
and v SNARE to interact1.
1-Sacher M. 2008. Traffic /2- Stephanie Brunet poster 2010 / 3- The cell, fifth edition,chapter 10, figure 10.35 /4-Ben-Aroya 2008,cell
Ref. 3Ref. 3
A high copy number screen of the trs23Δ99C mutant
Ref 2
C terminus
deletion
Triple
mutation
Cells were
still viable
P X
Reduces
GEF activity
P P
Destabilize
d TRAPPI
P NA
XV