MECHANISMS OF LOCALIZATION OF THE MOLECULAR CHAPERONE COSMC TO THE ENDOPLASMIC RETICULUM Open Access

Sun, Qian (2010)

Permanent URL: https://etd.library.emory.edu/concern/etds/9306sz92t?locale=en
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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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