Cargo-dependent Recruitment of AP-1 and GGA Adaptors to the Golgi, the TGN, and Recycling Endosomes Open Access

Caster, Amanda (2013)

Permanent URL: https://etd.library.emory.edu/concern/etds/nz806038c?locale=en
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Abstract

Membrane traffic requires the specific concentration of protein cargos into nascent carriers. Critical components of this selectivity are the protein adaptors that bind to short, linear motifs in the cytoplasmic tails of transmembrane protein cargos and sequester them into nascent carriers. My work examined the specificity and initial sites of recruitment of Arf-dependent adaptors (AP-1, GGAs, and Mint3) in response to the Golgi or endosomal localization of specific cargo proteins. Rigorous localization or co-localization data require quantitative image analyses that can vary widely between fields and laboratories. I developed a novel method for quantifying changes in signal intensity of one protein within any three-dimensional structure, defined by the presence of a different marker. I used the quantification of adaptor recruitment to cargos at the Golgi as examples of this method, though it can be directly applied to any site in the cell. I found that cargo promotes the recruitment of specific adaptors, suggesting that it is part of an upstream signaling event. I further discovered that AP-1 and GGAs are recruited to M6PR at recycling endosomes and not at the Golgi, as suggested by steady state staining profiles. I further demonstrated strict specificity for recruitment of the Mint3 adaptor by APP at the Golgi and identify LAMP1+ structures as the proximal destination of APP after leaving the Golgi. These results are discussed with respect to the generation of novel models for cargo-dependent regulation of membrane traffic.

Table of Contents

CHAPTER 1: Introduction and Background

1.1. The ARF Family of Regulatory GTPases.......................................................................2-6

1.1.A. ARF Evolution.......................................................................................................3

1.1.B. ARFs as Regulators of Critical Cellular Functions................................................4

1.1.C. ARGs as Regulators of Lipid Modifying Enzymes ...............................................6

1.2. ARF-dependent Adaptors at the Golgi ...........................................................................7-15

1.2.A. COPI......................................................................................................................7-9

1.2.B. Adaptins.................................................................................................................9-12

1.2.C. GGAs .....................................................................................................................12-14

1.2.D. Mints......................................................................................................................14-15

1.3. Cargo Proteins ................................................................................................................16-22

1.3.A. APP ......................................................................................................................16-17

1.3.B. Furin .....................................................................................................................18-20

1.3.C. M6PR ...................................................................................................................20-22

1.4. Guanine Nucleotide Exchange Factors ..........................................................................22-23

1.5. Key Concepts in Image Analysis....................................................................................23-24

1.6. Central Hypothesis .........................................................................................................24-39

CHAPTER 2: Computational Method for Calculating Fluorescence Intensities within Three- Dimensional Structures in Cells

2.1. Summary ........................................................................................................................41
2.2. Introduction ....................................................................................................................41-46

2.3. Results ............................................................................................................................46-54

2.4. 3D3I Applications...........................................................................................................54-65

2.5 Discussion .......................................................................................................................66

2.6.Experimental Procedures.................................................................................................66-70 2.7. Acknowledgements.........................................................................................................70-72

CHAPTER 3: A Role for Cargo in the Activation of ADP-Ribosylation Factors (Arf) and Adaptor Recruitment

3.1. Summary ........................................................................................................................74
3.2. Introduction ....................................................................................................................74-78

3.3. Results ............................................................................................................................79-109

3.4. Discussion ......................................................................................................................110-116

3.5. Experimental Procedures................................................................................................117-120

3.6. Acknowledgements.........................................................................................................120

CHAPTER 4: Recruitment of The Mint3 Adaptor Is Necessary For Export Of The Amyloid Precursor Protein (App) From The Golgi Complex

4.1. Summary ...................................................................................................................... 127
4.2. Introduction .................................................................................................................. 127-131

4.3. Results .......................................................................................................................... 131-152

4.4. Discussion .................................................................................................................... 153-158

4.5. Experimental Procedures.............................................................................................. 158-166

CHAPTER 5: Conclusions, Discussion, and Implications

5.1. Overview ...................................................................................................................... 168-169

5.2. ARF activation is a localized event that does not result in the presence of freely diffusing ARF

............................................................................................................................................. 169-171

5.3. Adaptors are recruited to specific membranes in a regulated fashion and in response to the presence of cargo at that site ............................................................................................... 171
5.4. Sorting motifs affect the traffic of cargo through the Golgi. ....................................... 172
5.5. Hypothesis #1 (Ch. 3): Fusion of GGA1/AP-1 mediated M6PR carriers originating at RE for retrieval to the Golgi is the slowest/rate-limiting step......................................................... 173-175

5.6. Hypothesis #2 (Ch. 3/4): Sorting motifs mediate traffic of cargo through the Golgi or function collect cargo at distinct compartments ................................................................................ 175-177

5.7. Hypothesis #3 (Ch. 4): LAMP1+ endosomes involved in the traffic of APP are non- degradative sorting stations ................................................................................................. 178-179

5.8. Concluding Remarks .................................................................................................... 179

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