Date of Award

Spring 1-1-2011

Document Type


Degree Name

Doctor of Philosophy (PhD)

First Advisor

Gia K. Voeltz

Second Advisor

Greg Odorizzi

Third Advisor

Andrew Staehelin

Fourth Advisor

Mark Winey

Fifth Advisor

Joseph Falke


The endoplasmic reticulum (ER) is an organelle that extends throughout the cell cytoplasm and has a complex membrane structure. There are three major ER domains, the nuclear envelope, the ER cisternae, and the tubular ER. Each domain is structured by its own set of membrane shaping proteins. The protein family that is the focus of this study is the reticulons. The reticulons generate curvature throughout the ER, specifically at the tubular ER and the edges of ER cisternae. All reticulons tested partition exclusively to high curvature ER and generate immobile oligomers. Every reticulon contains the reticulon homology domain (RHD) at its C-terminal end and this domain is sufficient for partitioning and oligomerizing in the high curvature ER. The RHD contains two short hairpin membrane domains (MD) that are predicted to generate membrane curvature by increasing the area of the cytoplasmic leaflet. In this study we test the functional contribution of the short hairpin MDs, in particular their length, in reticulon properties and function by generating mutants in human Rtn4 that had typical two-pass transmembrane domains (TMD), Rtn4TM. We tested the ability of Rtn4TM mutants to partition to tubules, form immobile oligomers, and reshape the peripheral ER. Our results show that Rtn4TM does not partition to tubules, cannot form immobile oligomers, and does not reshape ER like wild type Rtn4 showing that short hairpin MDs are important for reticulon function. Additionally, previous data shows that the longest mammalian reticulon isoform Rtn4a is phosphorylated at several residues within its unique ~80KDa N-terminal domain. We generated unphosphorylated and phosphomemetic mutants at serine residues 181, 182, and 184 in Rtn4a. We tested the phosphorylation mutants for localization, oligomerization, and ER shaping properties. We found that in interphase cells wild-type Rtn4a behaves similar to the phosphomemetic mutant. In contrast, the unphosphorylated mutant is even less mobile than Rtn4a and is unable to convert cisternae to tubules suggesting that Rtn4a function is regulated by phosphorylation.


Author also known as Agnes Zurek.