Document Type

Dissertation

Date of Degree

Spring 2015

Degree Name

PhD (Doctor of Philosophy)

Degree In

Biochemistry

First Advisor

Sheila A. Baker

Abstract

Photoreceptors are highly compartmentalized neurons in the retina, and they function by detecting light and initiating signaling through the visual network. The photoreceptor contains several compartments including the outer segment (OS) which is a sensory cilium for detecting photons and the inner segment (IS) that carries out important modulatory functions via its resident channels and transporters. Those proteins are membrane proteins that function together to shape electrical properties of the cell membrane during both rest and active states. Therefore it is essential to maintain proper function of the membrane proteins in the IS. One important way to regulate the function of a membrane protein is via controlling its trafficking to ensure a proper amount of the protein in the proper cellular compartment. To date, little is known about how IS membrane protein trafficking is controlled in photoreceptors. In this study, our goal is to understand those mechanisms using cell biology and biochemistry approaches. To achieve the goal, we investigated trafficking of two unrelated IS resident proteins: the hyperpolarization-activated cyclic nucleotide-gated channel 1 (HCN1) that mediates a feedback current in photoreceptors, and the sodium potassium ATPase (NKA) which maintains the basic electrochemical property of the cell.

In order to study trafficking of HCN1, we first investigated the dependence of HCN1 trafficking in photoreceptors on TRIP8b, an accessory subunit that influences trafficking of HCN1 in hippocampal neurons. By studying TRIP8b knockout mice we found that TRIP8b is dispensable for HCN1 trafficking in photoreceptors but required for maintaining the maximal expression level of HCN1. Since we revealed that HCN1 trafficking can be regulated in a cell-type specific manner, we subsequently focused on the amino acid sequence of HCN1 to identify novel trafficking signals that function in photoreceptors. By examining localization of a series of HCN1 mutants in transgenic Xenopus photoreceptors, we discovered a di-arginine ER retention motif and a leucine-based ER export motif. These two sequence motifs must function together to maintain equilibrium of HCN1 level between the endomembrane system and the cell surface. The study of HCN1 uncovered a mechanism for the photoreceptor to control membrane protein trafficking via the early secretory pathways.

To reveal additional trafficking machineries in photoreceptors, we investigated trafficking of NKA. We first tested for an interaction with ankyrin, an adaptor protein that regulates NKA trafficking in epithelial cells, and found these proteins do not co-localize in photoreceptors. We then aimed to identify novel trafficking signals by studying the trafficking behavior of two NKA isozymes: NKA-α 3 and NKA-α 4. When expressed in transgenic Xenopus photoreceptors, these two proteins localize to the IS and the OS respectively. By studying localization of multiple chimeras and truncation mutants, we found that the distinct localization pattern is due to a VxP OS/ciliary targeting motif present in NKA-α 4. Since NKA-α 4 is naturally expressed in the ciliary compartment of the sperm, our finding in the photoreceptor suggests a mechanism for NKA-alpha 4 trafficking in its native environment. Overall, our studies of HCN1 and NKA together provide new insights into controlling membrane protein trafficking in photoreceptors and help establish the basics for future therapeutic intervention targeting trafficking pathways that are linked to about one third of proteins reported in retinal diseases.

Public Abstract

Photoreceptors are neurons responsible for detecting light in the eyes. The inner segment (IS) is a specialized structure within a photoreceptor and contains several resident membrane proteins that function to control electrical properties of the cell membrane. The specialized localization of those proteins is controlled by protein transport mechanisms and is critical for functions of the proteins. Our goal is to understand how IS membrane proteins are transported in photoreceptors using cell biology and biochemistry approaches. To achieve this goal, we investigated trafficking of two unrelated IS proteins: the hyperpolarization-activated cyclic nucleotide-gated channel 1 (HCN1) that is required for fast light responses, and the sodium potassium ATPase (NKA) that shuttles ions across the cell membrane.

To study how HCN1 is transported, we first investigated the role of TRIP8b, a regulator of HCN1 transport in hippocampal neurons. Using genetically modified mice we found that TRIP8b is not required for proper HCN1 transport in photoreceptors. Using genetically modified frogs we subsequently identified two amino acid sequences within HCN1 itself that are required for transporting HCN1 from the inside of the cell to the cell surface. To study how NKA is transported, we studied transport of two NKA proteins that are transported to different places in the photoreceptor. Using genetically modified frogs we found that this different transport phenomenon is due to a ciliary transport sequence. Our findings provide new ideas in protein targeting and the basics for future therapeutic tools targeting protein transport mechanisms.

Keywords

publicabstract, HCN1, membrane protein, NKA, photoreceptor, trafficking, TRIP8b

Pages

xv, 142 pages

Bibliography

Includes bibliographical references (pages 126-142).

Copyright

Copyright 2015 Yuan Pan

Included in

Biochemistry Commons

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