Mechanism of Trafficking of Integral Proteins from the Endoplasmic Reticulum to the Inner Nuclear Membrane
(Dr. Sharon Braunagel, Shawn Williamson and Dr. Zhenping Zhong)

Current Research Projects:

Sharon Braunagel

Mutations within a number of resident inner nuclear membrane (INM) proteins are known to cause human diseases, including several types of muscular and lipid dystrophies.  Knowledge of function, protein and lipid composition, mechanisms of protein sorting, and trafficking to the INM are not well defined.  It is proposed that resident integral membrane proteins traffic to the INM by diffusion-retention.  By this model, INM integral membrane proteins freely diffuse between the endoplasmic reticulum (ER), outer nuclear membrane (ONM) and INM.  Interactions with nucleoplasmic components anchor these proteins at the INM, thus removing them from the diffusible pool.  While this model fits basic characteristics of some INM proteins, it does not explain enrichment at the INM of other resident proteins.  Recent studies have increased our knowledge from less than 10 to more than 67 INM proteins.  For some of these, the retention domain can be removed and the truncated protein still enriches at the INM.  Other INM proteins do not contain a discernable retention domain.  Thus it is our hypothesis that diffusion/retention is not the only mechanism for protein sorting, trafficking and enrichment at the INM.
Many viral processes mimic cellular mechanisms and pathways.  This feature, along with the property of viruses to provide a synchronous, amplified pulse of unique viral integral membrane proteins trafficking to specific organelles has resulted in kinetic descriptions and detailed knowledge of many aspects of cellular protein trafficking.   Baculovirus provides an amplified pulse of viral envelope proteins which transit from their site of insertion at the ER to the ONM and INM, and finally to membrane vesicles that form within the infected cell nucleus.  Thus, this virus provides a powerful tool to study the pathway of integral membrane proteins to the INM.  These same viral proteins are enriched at the nuclear envelope and INM in the absence of infection in both insect and mammalian cells.  This suggests that the mechanism(s) that regulates trafficking to the INM are not unique to the virus.

Specific Aims:
The long-range goal is to understand the mechanism of integral membrane protein sorting and trafficking to the INM. The goal of this project is to discern the mechanism utilized by baculovirus envelope proteins as they traverse the INM on their way to viral-induced membranes within the nucleus.  At every stage we will compare the mechanism(s) utilized by viral membrane proteins with mammalian INM proteins.  Thus, both viral and cellular INM protein markers will be utilized to test the hypothesis that more than one mechanism functions during trafficking to the INM. It is our expectation that significant insights on the biochemistry of protein trafficking to the INM will be revealed during these studies.

The integral membrane protein ODV-E66 is the primary viral marker used throughout this study.  This was chosen because a minimal amino acid sequence (33 amino acids) sufficient for trafficking to the ODV envelope has been identified within this protein.  This minimal sequence has distinctive characteristics that are present on both viral envelope and mammalian INM proteins; these features are named the Signature Motif (SM).  The mammalian resident INM proteins lamin B receptor (LBR) and nurim have been chosen for comparison to the viral marker. LBR, the most characterized INM protein, is representative of a larger group of INM proteins. Nurim represents a second class of INM proteins, with features significantly different from those represented by LBR.

Research Plan:
Our experimental strategy is to use the viral protein ODV-E66 and SM-fusions to investigate potential sites of regulation in the pathway to the nuclear membrane.  Special attention is directed to the first putative sites of regulation: 1) sorting at the time of primary translocation across the endoplasmic reticulum; 2) specific transport from the ER to the outer nuclear membrane and; 3) potential interaction with proteins of the nuclear pore complex as the protein traverses from the ONM to the INM.  Our strategy involves cross-linking and identification of proteins in close proximity during these intermediate phases.  With identity of potential partners determined, mutational analysis will be used to determine if the putative partners function during the trafficking process.

Shawn Williamson's PhD. dissertation study is focused on the integral membrane protein AcMNPV ODV-E66 (E66). This protein is localizes in the envelope of the occluded form of Autographa californica multinucleopolyhedrosis virus, a baculovirus.  During infection, this protein localizes to viral induced nuclear membrane structures termed microvesicles.  My research project is focused on localization of proteins to the nuclear membrane via a putative signature motif positioned at the N-terminus of E66. Previous research in this lab identified the N-terminal region of E66 was sufficient to localize reporter proteins, GFP and B-galactosidase, to virally induced microvesicles during infection.  More recent research has shown this sequence is sufficient for localization of EGFP at the nuclear envelope in the absence of infection.  The amino acid sequence of the motif shares characteristics with other nuclear integral membrane proteins including Emerin and Lamin B receptor and gB1 of Herpes Simplex Virus.  The goal of the project is to define what specific characteristics of the signature motif are required for trafficking, as well as to determine if and what molecular interactions are required to localize membrane proteins containing this motif to the nuclear envelope and viral induced nuclear microvesicles.  Briefly, I plan to do a mutational analysis of the signature motif by generating various constructs that alter the overall character of the motif.  Each of the different variants will be fused to EGFP and/or other fusion proteins and cloned into the appropriate vectors for transient expression in both insects and mammalian cells, as well as vectors required for recombinant virus.  Transfected and infected cells will be analyzed using light confocal microscopy(LCM), fluorescence recovery after photobleaching (FRAP), transmission electron microscopy (TEM) and biochemical assays. Additionally in vitro techniques including in vitro transcription and in vitro translation will be utilized.