MECHANISMS OF LOCALIZATION OF THE MOLECULAR CHAPERONE COSMC TO THE ENDOPLASMIC RETICULUM Pubblico
Sun, Qian (2010)
Abstract
Human Cosmc (Core 1 β3-Gal-T-Specific Molecular Chaperone)
is encoded by a single
exon gene on Xq24 and its cDNA predicts a 318-amino-acid
transmembrane glycoprotein
with type II topology. Cosmc acts as a specific molecular chaperone
for core 1 β3-
galactosyltransferase (C1β3Gal-T or T-synthase) and assists in
the folding/maturation of
this enzyme. Mutation in Cosmc accounts for the defects in
T-synthase, which is related
to some autoimmune diseases as well as some human cancers. Our
previous study
showed that Cosmc is primarily localized in the endoplasmic
reticulum (ER). Because
Cosmc does not have traditional ER retention or retrieval motifs,
the ER localization of
Cosmc is likely related to other structural features of the
protein. Here we explore the
potential retention/retrieval mechanism of Cosmc in the ER. In
order to address the role
of different domains of Cosmc on ER localization, six different
chimeric proteins were
generated and expressed in COS-7 cells, which allowed us to examine
the importance of
each domain in the ER localization. By co-localization with
intracellular markers, we
were able to determine that the transmembrane domain of Cosmc is
both necessary and
sufficient for its ER localization.
Table of Contents
Chapter 1
Introduction......................................................................................................1
1.1 Overview of the secretory pathway
...............................................................................1
1.2 Molecualr chaperones
....................................................................................................3
1.3 Background on protein glycosylation
............................................................................5
1.4 Background on
glycosyltransferases..............................................................................8
1.5 Background of T-synthase
...........................................................................................10
1.6 Backgound on Cosmc, a specific molecular chaperone for
T-synthase ......................11
1.7 Background on ER retention/ retrieval of proteins
......................................................13
Chapter 2 ER-localization determinants of Cosmc
......................................................18
2.1
Introduction..................................................................................................................18
2.2 Materials and
Methods.................................................................................................23
2.2.1
Reagents.............................................................................................................23
2.2.2 Preparation of expression constructs
.................................................................24
2.2.3 Site-directed mutagenesis
..................................................................................27
2.2.4 Cell culture and
transfection..............................................................................28
2.2.5 Immunoflurescent staining of COS7
cells.........................................................28
2.2.6 Subcellular
fractionation....................................................................................29
2.2.7 Preparation of cell extracts
................................................................................30
2.2.8 Western Blot
......................................................................................................30
2.2.9 Proteasome inhibitor
treatment..........................................................................31
2.2.10 In vitro Cosmc
immunoprecipitation...............................................................31
2.3
Results..........................................................................................................................31
2.3.1 Generation of Cosmc-TfR
chimeras..................................................................31
2.3.2 Wild type Cosmc localizes in the ER
................................................................33
2.3.3 Wild type TfR localizes in the plasma
membrane.............................................34
2.3.4 Cosmc/TfR/TfR (Construct #2) localizes in the plasma
membrane..................36
2.3.5 TfR/TfR/Cosmc (Construct #3) localizes in the
Golgi......................................38
2.3.6 Cosmc/Cosmc/TfR (Construct #4) localizes in the ER
.....................................40
2.3.7 TfR/Cosmc/TfR (Construct #5) localizes in the
ER..........................................42
2.3.8 Cosmc/TfR/Cosmc (Construct #6) mainly localizes in the Golgi,
but partially
in the ER
.........................................................................................................43
2.3.9 TfR/Cosmc/Cosmc (Construct #1) is unstable and degraded in
the proteasomes
..........................................................................................................................46
2.3.10 The amino acid cysteine within the membrane spanning domain
of Cosmc is
required for the retention of the full-length Cosmc in the ER
.........................47
2.4
Discussion....................................................................................................................49
Chapter 3 Summary and future directions
...................................................................54
References.........................................................................................................................57
List of Figures
Figure 1 Schematic diagram of transport steps between
membrane-bound organelles in a
typical eukaryotic cell
...........................................................................................2
Figure 2 Schematic diagram of catalytic function of T-synthase in
regulating biosynthesis
of T
antigen.........................................................................................................11
Figure 3 Working model for Cosmc function as an ER-localized
molecular chaperone
preventing aggregation/ proteasomal degradation of T-synthase
.......................13
Figure 4 Comparison of amino acid sequences of wild type Cosmc from
different species
..........................................................................................................................20
Figure 5 Schematic diagram of the PCR-mediated recombination
strategy used in making
chimeric constructs
.............................................................................................27
Figure 6 Schematic diagram of the subcellular fractionation
............................................29
Figure 7 Schematic diagram of the starting proteins and the
generated various chimaras
used in this
study.................................................................................................32
Figure 8 Localization of the
wtCosmc...............................................................................33
Figure 9 Localization of the wtTfR
...................................................................................35
Figure 10 Localization of the construct Cosmc/TfR/TfR
..................................................37
Figure 11 Localization of the construct TfR/TfR/Cosmc
..................................................39
Figure 12 Localization of the construct Cosmc/Cosmc/TfR
.............................................41
Figure 13 Localization of the construct TfR/Cosmc/TfR
..................................................43
Figure 14 Localization of the construct Cosmc/TfR/Cosmc
.............................................45
Figure 15 Degradation of the construct TfR/Cosmc/Cosmc through
proteasome pathway.....47
Figure 16 Localization of the Cysteine mutant
Cosmc......................................................48
Figure 17 Summary of the
results......................................................................................50
List of Tables
Table 1 PCR primers used for making chimeric constructs
..............................................24
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