Molecular origin of plasma membrane citrate transporter in human prostate epithelial cells.
The prostate is a highly specialized mammalian organ that produces and releases large amounts of citrate. However, the citrate release mechanism is not known. Here, we present the results of molecular cloning of a citrate transporter from human normal prostate epithelial PNT2-C2 cells shown previously to express such a mechanism. By using rapid amplification of cDNA ends PCR, we determined that the prostatic carrier is an isoform of the mitochondrial transporter SLC25A1 with a different first exon. We confirmed the functionality of the clone by expressing it in human embryonic kidney cells and performing radiotracer experiments and whole-cell patch-clamp recordings. By using short interfering RNAs targeting different parts of the sequence, we confirmed that the cloned protein is the main prostatic transporter responsible for citrate release. We also produced a specific antibody and localized the cloned transporter protein to the plasma membrane of the cells. By using the same antibody, we have shown that the cloned transporter is expressed in non-malignant human tissues.
Mazurek
Maciej P
MP
Division of Cell and Molecular Biology, Sir Alexander Fleming Building, Imperial College London, South Kensington Campus, London SW7 2AZ, UK.
Prasad
Puttur D
PD
Gopal
Elangovan
E
Fraser
Scott P
SP
Bolt
Leszek
L
Rizaner
Nahit
N
Palmer
Christopher P
CP
Foster
Christopher S
CS
Palmieri
Ferdinando
F
Ganapathy
Vadivel
V
Stühmer
Walter
W
Djamgoz
Mustafa B A
MB
Mycielska
Maria E
ME
D000595:Amino Acid Sequence; D027321:Anion Transport Proteins / Q000737:chemistry / Q000378:metabolism; D001692:Biological Transport; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D019343:Citric Acid / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D019143:Evolution, Molecular; D020868:Gene Silencing; D006801:Humans; D007150:Immunohistochemistry; D007477:Ions / Q000378:metabolism; D008297:Male; D024101:Mitochondrial Proteins / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D027361:Organic Anion Transporters; D011467:Prostate / Q000166:cytology*; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism
D027321:Anion Transport Proteins; D002352:Carrier Proteins; D007477:Ions; D024101:Mitochondrial Proteins; D027361:Organic Anion Transporters; D020033:Protein Isoforms; C532592:Slc25a1 protein, human; C020723:citrate-binding transport protein; D019343:Citric Acid
DNA Seq. 1997;7(3-4):127-39
9254007
J Biol Chem. 2002 Oct 18;277(42):39469-76
12177002
Horm Metab Res. 2002 Aug;34(8):417-24
12198595
Eur J Radiol. 2007 Sep;63(3):351-60
17709223
Biochim Biophys Acta. 1989 Nov 23;977(2):171-6
2804096
Biochim Biophys Acta. 1993 Apr 5;1142(1-2):139-45
8457581
Clin Cancer Res. 2005 Aug 1;11(15):5381-9
16061851
Prostate. 1994;24(3):139-42
8115279
Biochem J. 2009 Apr 15;419(2):369-75
19143589
Mol Reprod Dev. 2006 Mar;73(3):369-78
16362974
Plant Physiol. 1999 Dec;121(4):1375-82
10594125
J Physiol. 2005 Mar 1;563(Pt 2):393-408
15611019
Breast Cancer Res Treat. 2007 Jan;101(2):149-60
16838113
J Physiol. 2004 Sep 15;559(Pt 3):821-33
15254154
FEBS Lett. 1995 Jan 9;357(3):297-300
7835431