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1.  Proteomic analysis of the cilia membrane of Paramecium tetraurelia 
Journal of proteomics  2012;78:113-122.
Channels, pumps, receptors, cyclases and other membrane proteins modulate the motility and sensory function of cilia, but these proteins are generally under-represented in proteomic analyses of cilia. Studies of these ciliary membrane proteins would benefit from a protocol to greatly enrich for integral and lipidated membrane proteins. We used LC-MS/MS to compare the proteomes of unfractionated cilia (C), the ciliary membrane (CM) and the ciliary membrane in the detergent phase (DP) of Triton X-114 phase separation. 55% of the proteins in DP were membrane proteins (i.e. predicted transmembrane or membrane-associated through lipid modifications) and 31% were transmembrane. This is to be compared to 23% membrane proteins with 9% transmembrane in CM and 9% membrane proteins with 3% transmembrane in C. 78% of the transmembrane proteins in the DP were found uniquely in DP, and not in C or CM. There were ion channels, cyclases, plasma membrane pumps, Ca2+ dependent protein kinases, and Rab GTPases involved in the signal transduction in DP that were not identified in the other C and CM preparations. Of 267 proteins unique to the DP, 147 were novel, i.e. not found in other proteomic and genomic studies of cilia.
PMCID: PMC3667161  PMID: 23146917
Ciliary membrane; transmembrane proteins; lapidated proteins; Triton X-114 phase separation; detergent phase; MS analysis
2.  Role of Plasma Membrane Calcium ATPases in Calcium Clearance from Olfactory Sensory Neurons 
Chemical Senses  2009;34(4):349-358.
Odorants cause Ca2+ to rise in olfactory sensory neurons (OSNs) first within the ciliary compartment, then in the dendritic knob, and finally in the cell body. Ca2+ not only excites but also produces negative feedback on the transduction pathway. To relieve this Ca2+-dependent adaptation, Ca2+ must be cleared from the cilia and dendritic knob by mechanisms that are not well understood. This work focuses on the roles of plasma membrane calcium pumps (PMCAs) through the use of inhibitors and mice missing 1 of the 4 PMCA isoforms (PMCA2). We demonstrate a significant contribution of PMCAs in addition to contributions of the Na+/Ca2+ exchanger and endoplasmic reticulum (ER) calcium pump to the rate of calcium clearance after OSN stimulation. PMCAs in neurons can shape the Ca2+ signal. We discuss the contributions of the specific PMCA isoforms to the shape of the Ca2+ transient that controls signaling and adaptation in OSNs.
PMCID: PMC2671884  PMID: 19304763
calcium; kinetics; mouse; olfactory neurons; PMCA; pumps
4.  Individuals with mutations in XPNPEP3, which encodes a mitochondrial protein, develop a nephronophthisis-like nephropathy  
The autosomal recessive kidney disease nephronophthisis (NPHP) constitutes the most frequent genetic cause of terminal renal failure in the first 3 decades of life. Ten causative genes (NPHP1–NPHP9 and NPHP11), whose products localize to the primary cilia-centrosome complex, support the unifying concept that cystic kidney diseases are “ciliopathies”. Using genome-wide homozygosity mapping, we report here what we believe to be a new locus (NPHP-like 1 [NPHPL1]) for an NPHP-like nephropathy. In 2 families with an NPHP-like phenotype, we detected homozygous frameshift and splice-site mutations, respectively, in the X-prolyl aminopeptidase 3 (XPNPEP3) gene. In contrast to all known NPHP proteins, XPNPEP3 localizes to mitochondria of renal cells. However, in vivo analyses also revealed a likely cilia-related function; suppression of zebrafish xpnpep3 phenocopied the developmental phenotypes of ciliopathy morphants, and this effect was rescued by human XPNPEP3 that was devoid of a mitochondrial localization signal. Consistent with a role for XPNPEP3 in ciliary function, several ciliary cystogenic proteins were found to be XPNPEP3 substrates, for which resistance to N-terminal proline cleavage resulted in attenuated protein function in vivo in zebrafish. Our data highlight an emerging link between mitochondria and ciliary dysfunction, and suggest that further understanding the enzymatic activity and substrates of XPNPEP3 will illuminate novel cystogenic pathways.
PMCID: PMC2827951  PMID: 20179356
5.  Plasma Membrane Calcium Pumps in Mouse Olfactory Sensory Neurons 
Chemical senses  2006;31(8):725-730.
We report here the presence of specific plasma membrane calcium pumps (PMCAs) in mouse olfactory sensory neurons. All 4 isoforms are present as shown by deconvolution microscopy, and the specific splice variants are identified by reverse transcriptase (RT)–polymerase chain reaction (PCR). The PMCAs are present on the cell body, dendrite, knob, and cilia, but the different isoforms of PMCAs are not identical in their distributions. The PMCAs are positioned to play a role in calcium clearance after stimulation.
PMCID: PMC2442823  PMID: 16855061
calcium; mouse; olfaction; pumps
6.  Glycosyl Phosphatidylinositol-Anchored Proteins in Chemosensory Signaling: Antisense Manipulation of Paramecium tetraurelia PIG-A Gene Expression†  
Eukaryotic Cell  2003;2(6):1211-1219.
Glycosyl phosphatidylinositol (GPI)-anchored proteins are peripheral membrane proteins tethered to the cell through a lipid anchor. GPI-anchored proteins serve many functions in cellular physiology and cell signaling. The PIG-A gene codes for one of the enzymes of a complex that catalyzes the first step in anchor synthesis, and we have cloned the Paramecium tetraurelia pPIG-A gene using homology PCR. To understand the function of pPIG-A and the significance of GPI-anchored proteins in Paramecium, we reduced the mRNA for pPIG-A in transformed cells using an expression vector that transcribed antisense mRNA. The amount of transcript is reduced to ∼0.3% of the mRNA in control-transformed cells. Compared to control cells, cells transformed with the antisense pPIG-A vector show reduced synthesis of GPI anchor intermediates catalyzed in their endoplasmic reticula and a very few GPI-anchored proteins among the peripheral proteins that can be recovered from their surfaces. They also show specific defects in chemoresponse to glutamate and folate. Other cellular functions, such as growth and mating, seem to be normal.
PMCID: PMC326658  PMID: 14665456

Results 1-6 (6)