Technical Reference #1674

1674.

The Intracellular Mobility of NPY and a Putative Mitochondrial form of NPY in Neuronal Cells Katja Kaipio; Ullamari Pesonen, University of Turku, Neuroscience Letters, 450(1674), (2009)

Abstract:
Preproneuropeptide Y is a precursor peptide to mature neuropeptide Y (NPY) which is a universally expressed peptide in the central and peripheral nervous system. NPY is normally routed to endoplasmic reticulum and secretory vesicles in cells which secrete NPY. In our previous studieswe found a functional Leucine7 to Proline7 (L7P) polymorphism in the signal peptide sequence of preproNPY. This polymorphism affects the secretion of NPY and causes multiple physiological effects in humans. The sequence of NPY mRNA contains two in frame kozak sequences that allow translation initiation to shift and translation of two proteins. In addition to mature NPY1–36 also a putative truncated NPY17–36 with mitochondrial targeting signal is produced. The purpose of this study was to investigate the protein mobility of the putative mitochondrial fragment and the effect of the L7P polymorphism on the cellular level using GFP tagged constructs. The mobility was studied with fluorescence recovery after photobleaching technique in a neuronal cell line.We found that the mobility of the secretory vesicles with NPY1–36 in cells with L7P genotype was increased in comparison to vesicle mobility in cells with the more abundant L7L genotype. The mobility in the cells with the putative mitochondrial construct was found to be very low. According to the results of the present study the mitochondrial truncated peptide stays in the mitochondrion. It can be hypothesized that this could be one of the factors affecting energy balance of the membranes of the mitochondrion.

Keywords:
Neuropeptide Y; leucine 7 to proline7 polymorphism; kozak sequence; mitochindria; endoplasmic reticulum; photobleaching

Materials & Methods:
Constructs and cell culture The constructs were prepared as described previously [10]. In the present study four different constructswere examined(Table 1) in neuronal SK-N-BE cell line (ATCC Manassas VA USA). The transfections and cell culture were done as previously described [10] under neomycin selection (50 mg/ml; Sigma St. Louis MO USA) in 25cm2 cell culture flasks. Formicroscopic experiments the cells were plated on a 35mmglass-bottomed culture dishes with 10mm microwell (MatTek Ashland MA USA).

Microscopic Technique
Confocal Microscopy

Cell Type(s)
Neurons