Semester

Fall

Date of Graduation

2011

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Neurology

Committee Chair

Visvanathan Ramamurthy

Abstract

Prenylation is a posttranslational modification which adds a lipid, either a farnesyl or geranylgeranyl group, to the C-terminal cysteine of proteins ending in a "CAAX" motif. Two additional steps referred to as "CAAX" protein processing follow prenylation. First, the terminal three amino acids are cleaved by the endoprotease RAS converting enzyme1 (RCE1), then isoprenylcysteine methyltransferase (ICMT) adds a methyl group to the cysteine. While it is clear that the addition of the prenyl group is required for the association of proteins with membranes, the function of proteolysis and methylation is not obvious. Two lines of evidence suggest a critical role for "CAAX" protein processing, particularly in neurons. First, the brain has the highest methyltransferase activity of all tissues tested and second, mice lacking RCE1 or ICMT are embryonic lethal. The objective of this study is to investigate the importance of "CAAX" protein processing in a neuronal system. The two enzymes responsible for the processing steps were genetically eliminated from the developing retina to evaluate the role of proteolysis and methylation in the stability, assembly, and localization of retinal proteins.;Retinas lacking Rce1 were morphologically similar to littermate controls at early ages. However, by the time the mice open their eyes there is a reduction in the number of photoreceptor cell nuclei which continues until the entire photoreceptor cell layer has degenerated by adulthood. Degeneration is exclusive to photoreceptor cells as other downstream neurons in the retina do not exhibit any signs of cell death in the absence of RCE1-mediated endoproteolysis. The rod and cone photoreceptor neurons remaining at post-natal day (P) 14 do not function as measured by electroretinography (ERG). Our studies also reveal that phosphodiesterase 6 (PDE6), a prenylated heteromeric protein cannot be transported to the outer segment (OS) in the absence of Rce1. This is in contrast to other prenylated proteins, such as the G-protein transducin or rhodopsin kinase, which are transported normally. Our studies conclusively demonstrate that RCE1-mediated endoproteolysis is critical for proper functioning and stability of photoreceptor neurons and more specifically it is needed for transport of PDE6.;In contrast, retinas lacking Icmt showed much more subtle defects than those observed upon elimination of Rce1. Elimination of Icmt message was confirmed by qPCR. To our surprise, no developmental defects were observed even though Six3-Cre eliminates Icmt at early embryonic ages. The earliest noted defect was a reduction in the rod photoreceptor response as measured by ERG. Loss of rod function is likely a consequence of reduction of prenylated phototransduction proteins, PDE6, GRK1 and most dramatically, transducin. Transducin gamma-subunit levels were reduced by 90% in animals lacking Icmt. Interestingly, PDE6 reduction was not associated with a trafficking defect as observed in the conditional knockout of Rce1 in the retina.;These are the first direct investigations of "CAAX" protein processing in an in vivo neuronal system. This is significant because neurons are known to contain a large population of unknown methylated proteins and express "CAAX" processing enzymes at high levels. Our studies are the first to show that RCE1-mediated proteolysis is required for transport of PDE6. In contrast, studies using the conditional knockout of Icmt demonstrate that PDE6 does not require methyl esterification for transport. Our findings suggest the presence of a unique PDE6 transport pathway that is distinct from previously characterized phototransduction protein transport pathways. Another contribution of this research has been to demonstrate that the PDE6 holoenzyme is assembled in the inner segment of photoreceptor cells. Altogether, the results add to our basic understanding of the contributions of posttranslational modifications of proteins to cellular function.

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