Progress 10/01/02 to 09/30/05
Outputs
We developed methods to purify a phospholipid flippase from the cytoplasmic membrane of Mycoplasma bovis. These methods led to a fraction highly enriched in flippase activity that contained four major proteins. Sequence information was obtained on one of these proteins. Efforts to obtain sequence for the other protein by peptide fingerprinting through mass spectrometry were unsuccessful, primarily because the M. bovis genome has not been sequenced. In view of this we diverted our efforts to flippase purification from sequenced organisms. This work is described under project number WIS03750. Our Mycoplasma work is described in a preprint that has not been published.
Impacts
This work has fundamental impact on our understanding of membrane bilayer assembly in cells.
Publications
- Transport of newly synthesized sterol to the sterol-enriched plasma membrane occurs via nonvesicular equilibration.Biochemistry. 2005 Apr 19;44(15):5816-26.PMID: 15823040 [PubMed - indexed for MEDLINE]
- Effects of conformational stability and geometry of guanidinium display on cell entry by beta-peptides.J Am Chem Soc. 2005 Mar 23;127(11):3686-7. No abstract available.PMID: 15771489 [PubMed - indexed for MEDLINE]
- Endoplasmic reticulum localization of Gaa1 and PIG-T, subunits of the glycosylphosphatidylinositol transamidase complex.J Biol Chem. 2005 Apr 22;280(16):16402-9. Epub 2005 Feb 15.PMID: 15713669 [PubMed - indexed for MEDLINE]
- Flip-flop of glycosylphosphatidylinositols (GPI's) across the ER. Chem Commun (Camb). 2005 Jan 28;(4):453-5. Epub 2004 Dec 2. PMID: 15654367 [PubMed - in process]
|
Progress 01/01/04 to 12/31/04
Outputs
We developed methods to purify a phospholipid flippase from the cytoplasmic membrane of Mycoplasma bovis. These methods led to a fraction highly enriched in flippase activity that contained four major proteins. Sequence information was obtained on one of these proteins. Efforts to obtain sequence for the other protein by peptide fingerprinting through mass spectrometry were unsuccessful, primarily because the M. bovis genome has not been sequenced. In view of this we diverted our efforts to flippase purification from sequenced organisms. This work is described under project number WIS03750. Our Mycoplasma work is described in a preprint that has not been published.
Impacts
This work has fundamental impact on our understanding of membrane bilayer assembly in cells.
Publications
- No publications reported this period
|
Progress 01/01/03 to 12/31/03
Outputs
Phospholipid flip-flop in biogenic (self-synthesizing) membranes is a facilitated diffusion process essential for membrane bilayer propagation. Although over 25 years have elapsed since it was first hypothesized that specific proteins, flippases, are responsible for facilitating flip-flop, a phospholipid flippase has yet to be identified. In order to address this problem, we selected Mycoplasmas as a model system since these simple bacterial cells have a single biogenic membrane and a small genome that encodes only 100 membrane proteins. We hypothesized that one of these membrane proteins would be a phospholipid flippase and that Mycoplasma membranes would represent an ideal source from which to isolate this essential transporter. In support of the hypothesis we demonstrate that proteoliposomes reconstituted from a Triton X-100 extract of salt-stripped membranes of Mycoplasma bovis are capable of facilitating flop of fluorescent phospholipids using two independent
assays. Protease treatment of the proteoliposomes abrogated translocation, and protein-free vesicles prepared with M. bovis lipids were inactive. We calculate that active flippases represent 1% by mass of M. bovis membrane proteins in the detergent extract. Using three chromatographic steps we fractionated the detergent extract to generate material 40-fold enriched in flippase activity, containing three major low molecular weight flippase candidates separated on Coomassie-stained SDS-PAGE. Our results (i) indicate that flippase activity is embodied in specific proteins, (ii) suggest that a phospholipid flippase should be included in the roster of membrane proteins coded for by a minimal genome and (iii) set the stage for identification and characterization of a phospholipid flippase protein.
Impacts
This work has fundamental impact on our understanding of membrane bilayer assembly in cells.
Publications
- Watkins, W.E., Thomas, C.B., Walent, J.H. & Menon, A.K. (2003) Partial purification of a phospholipid flippase from the minimal proteome of Mycoplasma bovis. Eur. J. Biochem. in revision.
|
Progress 01/01/02 to 12/31/02
Outputs
Glycerophospholipids are synthesized on the cytoplasmic face of biogenic membranes and initially located in the cytoplasmic leaflet of the bilayer. The lipids must translocate across the bilayer to populate the exoplasmic leaflet for membrane assembly. We recently completed studies, based on a detergent-solubilization-biochemical-reconstitution approach, that showed that specific proteins in mammalian ER and bacterial cytoplasmic membranes catalyze flip-flop. We have extended these studies to develop assays using natural phospholipids (not phospholipid analogs as used previously), and to identify flippase-enriched protein fractions from detergent extracts of bacterial or endoplasmic reticulum. We have further extended our studies to include analyses of Mycoplasma bovis, an organism with a limited repertoire of membrane proteins. An abstract of our work on mycoplasmas is reproduced here: The transbilayer diffusion (flip-flop) of phospholipids in biogenic
(self-synthesizing) membranes is a bidirectional, metabolic-energy-independent, protein-mediated process critical to the process of membrane bilayer assembly. The phospholipid translocator or flippase responsible for facilitating flip-flop has yet to be identified. In order to make progress towards identifying a flippase and to test whether a flippase is likely to be part of the minimal equipment required for cellular life, we chose to work with mycoplasmas. These microbes have small genomes that closely resemble the minimal genome predicted to be required for cellular life; they consequently have relatively few membrane proteins (100) making them a good choice for the purification of a novel transporter. We developed a membrane reconstitution procedure in which detergent extracts of salt-stripped membranes of Mycoplasma bovis were reconstituted into proteoliposomes capable of facilitating the transbilayer movement of fluorescent phospholipid analogues using two independent assays.
Protease treatment abrogated the ability of the proteoliposomes to translocate lipids, and vesicles prepared with M. bovis lipids were inactive. Our data suggest that active flippases represent 1% by mass of the M. bovis membrane proteins present in the detergent extract. Chromatographic resolution of the M. bovis detergent-extract yielded a fraction enriched in flippase activity that contained four major proteins as visualized by SDS-PAGE and Coomassie staining. Our results (i) set the stage for the eventual identification of a flippase and an understanding of the molecular mechanism of transport and (ii) suggest that a phospholipid flippase should be included in the roster of membrane proteins coded for by a minimal genome.
Impacts
The project will yield fundamental new information on how cell membranes are assembled.
Publications
- No publications reported this period
|
|