Date of Award

2015

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Cell & Molecular Biology

First Advisor

Michael S. Marks

Abstract

Melanosomes are subcellular organelles specialized for the synthesis and storage of melanin pigments. Within epidermal and ocular pigment cells, melanosomes are generated from endosomal precursors in discrete stages. Early stages are characterized by the accumulation of premelanosome protein (PMEL) and its assembly into nontoxic amyloid fibrils, while later stages are characterized by the arrival of proteins important for the production of pigment. In this thesis, I will discuss two important findings relevant to melanosome biology.

The first finding addresses the molecular mechanisms that regulate the transformation of PMEL from an integral membrane protein to fibrillar structures with properties of amyloid. These data show that native disulfide-bonded PMEL dimers prevent premature fibril formation early in the secretory pathway but must be resolved prior to the assembly of functional amyloid fibrils in early stage melanosomes. Failure to resolve the dimeric intermediates – as occurs with mutagenesis of a PMEL regulatory domain – decreases amyloid production in a heterologous expression system. Since the oligomerization of amyloid precursors is hardly unique, detailed characterization of the different intermediates formed by functional versus pathological amyloid proteins may bring us one step closer to understanding the mechanisms of neurodegenerative amyloid disease.

The second finding addresses the localization and potential function of SLC45A2, a proton-dependent sucrose transporter encoded by the gene that is mutated in the hypopigmentary disorder oculocutaneous albinism type 4 (OCA4). While melanocytes derived from patients and mouse models of OCA4 are known to contain hypopigmented melanosomes, it is unclear why mutations in SLC45A2 lead to decreased pigment production. Our data show that SLC45A2 localizes to and functions from distinct punctate structures on/near stage III and IV melanosomes. Mislocalization of SLC45A2 leads to hypopigmentation suggesting that the transporter must be present on melanosomal structures to function. Preliminary data also suggest that SLC45A2-deficient melanocytes harbor melanosomes that are hypopigmented but normally shaped and that SLC45A2 function can be bypassed by overexpression of a different melanosomal transporter, OCA2. Along with other preliminary data, these results suggest that SLC45A2 likely maintains a neutral pH within maturing melanosomes to ensure sustained melanin deposition.

Together, these two studies provide insight into the molecular mechanisms that regulate nontoxic amyloid and melanin production within healthy melanocytes.

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