Flip-Flop of Phospholipids in Proteoliposomes Reconstituted from Detergent Extract of Chloroplast Membranes: Kinetics and Phospholipid Specificity

Eukaryotic cells are compartmentalized into distinct sub-cellular organelles by lipid bilayers, which are known to be involved in numerous cellular processes. The wide repertoire of lipids, synthesized in the biogenic membranes like the endoplasmic reticulum and bacterial cytoplasmic membranes are initially localized in the cytosolic leaflet and some of these lipids have to be translocated to the exoplasmic leaflet for membrane biogenesis and uniform growth. It is known that phospholipid (PL) translocation in biogenic membranes is mediated by specific membrane proteins which occur in a rapid, bi-directional fashion without metabolic energy requirement and with no specificity to PL head group. A recent study reported the existence of biogenic membrane flippases in plants and that the mechanism of plant membrane biogenesis was similar to that found in animals. In this study, we demonstrate for the first time ATP independent and ATP dependent flippase activity in chloroplast membranes of plants. For this, we generated proteoliposomes from Triton X-100 extract of intact chloroplast, envelope membrane and thylakoid isolated from spinach leaves and assayed for flippase activity using fluorescent labeled phospholipids. Half-life time of flipping was found to be 6±1 min. We also show that: (a) intact chloroplast and envelope membrane reconstituted proteoliposomes can flip fluorescent labeled analogs of phosphatidylcholine in ATP independent manner, (b) envelope membrane and thylakoid reconstituted proteoliposomes can flip phosphatidylglycerol in ATP dependent manner, (c) Biogenic membrane ATP independent PC flipping activity is protein mediated and (d) the kinetics of PC translocation gets affected differently upon treatment with protease and protein modifying reagents

A Fluorescent Glycolipid-Binding Peptide Probe Traces Cholesterol Dependent Microdomain-Derived Trafficking Pathways

10.1371/journal.pone.0002933PLoS ONE38

Looking at membrane lipids from the inside of the cell

A novel biosensor developed to visualize phosphatidylserine in intact cells suggests a new role for the anionic lipid in specifying intracellular membranes involved in signaling events

Immunohistochemical staining of human islet cells with region-specific antibodies against secretogranins II and III

Chromogranins and secretogranins belong to the granin family of proteins, which are expressed in neuroendocrine and nervous tissue. In earlier publications we have described the development of region-specific antibodies against CgA and CgB. In this study we describe antibodies to SgII and SgIII and their usefulness for immunohistochemical staining. Peptides homologous to defined parts of secretogranins II and III were selected and synthesized. Antibodies were raised and immunostainings were performed on normal human pancreas. The SgII 154–165 (N-terminal secretoneurin), SgII 172–186 (C-terminal secretoneurin) and SgIII antibodies immunostained all insulin-immunoreactive cells, most of the glucagon cells and some of the pancreatic polypeptide cells. The SgII 225–242 antibody immunostained only the insulin-containing cells. None of the antibodies immunostained the somatostatin cells. This study is the first observation of the expression of SgIII in human tissues, where we show expression of SgIII in three of the four major islet cell types in human pancreas

Swf1-dependent palmitoylation of the SNARE Tlg1 prevents its ubiquitination and degradation

Protein palmitoylation is a post-translational modification that affects a great number of proteins. In most cases, the enzymes responsible for this modification have not been identified. Some proteins use palmitoylation to attach themselves to membranes; however, palmitoylation also occurs in transmembrane proteins, and the function of this palmitoylation is less clear. Here we identify Swf1, a member of the DHHC-CDR family of palmitoyltransferases, as the protein responsible for modifying the yeast SNAREs Snc1, Syn8 and Tlg1, at cysteine residues close to the cytoplasmic end of their single transmembrane domains (TMDs). In an swf1Δ mutant, Tlg1 is mis-sorted to the vacuole. This occurs because unpalmitoylated Tlg1 is recognised by the ubiquitin ligase Tul1, resulting in its targeting to the multivesicular body pathway. Our results suggest that one role of palmitoylation is to protect TMDs from the cellular quality control machinery, and that Swf1 may be the enzyme responsible for most, if not all, TMD-associated palmitoylation in yeast