J Cell Biol. Sdhof, 1999 ). The mechanisms responsible for targeting these proteins specifically to the SV membrane are poorly understood. Integral membrane proteins are synthesized on the rough endoplasmic reticulum and traffic through the Golgi network. Proteins exiting the (AP180 homolog) mutant, synaptobrevin alone is mislocalized, whereas other SV proteins are unaffected (Nonet (Fergestad has several features that make it a valuable system for studying both K145 hydrochloride synaptic function and SV protein localization in vivo. Both forward and reverse genetics have been used to generate mutations in multiple synaptic components, many of which are evolutionarily conserved (Fernandez-Chacon and Sdhof, 1999 K145 hydrochloride ; Nonet, 1999 ). The unique life cycle of Sequencing Consortium, 1998 ). Qualitative and quantitative analysis of the distribution of GFP-tagged protein in live animals under fluorescent microscopy is simple owing to the transparent nature of the organism (Labrousse gene (Stenius member, has 30% identity to the rat synaptogyrin and a similar hydrophobicity profile (Nonet, 1999 ). Analysis of animals expressing green fluorescent protein (GFP)-tagged synaptogyrin (SNG-1) suggests that SNG-1 is localized to synaptic regions (Nonet, 1999 ). Thus far, no other synaptogyrin family members have been identified. We have identified two primary sequences in SNG-1 that are necessary for its synaptic localization, a C-terminal region containing 38 amino acids and an arginine in the loop facing the cytoplasm. An SNG-1 sequence containing these elements is sufficient to localize rat cellugyrin that is normally not restricted to synaptic regions in Furthermore, rat synaptogyrin is localized to synaptic regions in both and hippocampal neurons in culture and the C terminus is necessary for synaptic localization in each system. Our results suggest that the mechanisms used for synaptic localization among synaptogyrin family members are evolutionarily conserved. MATERIALS AND METHODS Nematode Strains and Culture Bristol strain N2, mutants, and transgenic animals were grown at 22.5C on solid medium as described by Rabbit Monoclonal to KSHV ORF8 Sulston and Hodgkin (1988) . Microscopy and Image Analysis Live animals were anesthetized with 10 mM sodium azide, mounted on 2% agarose pads, and examined under epifluorescence with the use of an BX60. Time-averaged images were collected with the use of a DAGE SIT68 camera and a CG-7 frame K145 hydrochloride grabber (Scion Image). Confocal images were taken in a series of 0.5 M optical sections with the use of an Fluoview microscope and Fluoview software. A three-dimensional representation of a 50-m segment of sublateral ventral nerve cord just anterior to the posterior end point of the cord near the tail (boxed area in Figure ?Figure4B)4B) was made for quantitative analysis. Total intensity in the boxed area was measured with the use of K145 hydrochloride Fluoview software. Background fluorescence was subtracted as the average intensity of areas adjacent to the boxed region. Total intensity in puncta (closed circles in Figure ?Figure4B),4B), which were defined as discrete bright fluorescent patches, was calculated by summing the intensity in each punctum. The percentage of fluorescence in puncta was calculated by dividing the total intensity in the puncta by the total intensity in the boxed area after background subtraction. Open in a separate window Figure 4 Fine mapping of SNG-1 localization signals. (A) Sequence alignment of SNG-1, rat synaptogyrin (rat-sgyr), and cellugyrin (rat-cgyr). Amino acids are represented by the standard one-letter code and identical amino acids are boxed. Shaded regions are transmembrane domains. Amino acids 173C210 are overlined. R104 is marked by asterisk. Y174, Y201, and Y203 are marked by closed circles. (B) Diagram of the.