Ubiquitin-activating enzyme (E1) isoforms in lens epithelial cells: origin of translation, E2 specificity and cellular localization determined with novel site-specific antibodies.
Lens development and response to peroxide stress are associated with dramatic changes in protein ubiquitination, reflecting dynamic changes in activity of the ubiquitin-activating enzyme (E1). Two isoforms of E1 (E1A and E1B) have been identified in lens cells although only one E1 mRNA, containing three potential translational start sites, has been detected. Novel, site-specific antibodies to E1 were generated and the hypothesis that the two isoforms of E1 are translated from alternative initiation codons of a single mRNA was tested. Antibodies raised against E1A-N peptide (Met(1)to Cys(23)of E1A) reacted only with E1A by immunoblot and immunoprecipitation. Antibodies raised against E1B-N peptide (Met(1)to Glu(25)of E1B or Met(41)to Glu(65)of E1A) and E1AB-C peptide (His(1030)to Arg(1058)of E1A or His(990)to Arg(1018)of E1B) reacted with both E1A and E1B. These results indicate that (1) E1A and E1B contain the same C-terminal residues; (2) E1A contains the N terminal sequence of E1B; and (3) E1B does not contain the N terminal sequence of E1A. The two isoforms of lens E1 are therefore translated from a single mRNA. Specifically, E1A is translated from the first initiation codon, and E1B translated from the second initiation codon. E1A and E1B were affinity-purified, and their ability to 'charge' ubiquitin carrier proteins (E2s) with activated ubiquitin was compared in a cell-free system. E1A and E1B were indistinguishable with respect to charging different E2s. However, E1 immunolocalization studies with human lens epithelial cells indicate that E1A and E1B are preferentially localized to the nucleus and cytosol, respectively. This observation suggests that E1A and E1B ubiquitinate different proteins and serve different functions in intact cells.
Shang
F
F
Laboratory for Nutrition and Vision Research, Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University, 711 Washington Street, Boston, MA 02111, USA.
Deng
G
G
Obin
M
M
Wu
C C
CC
Gong
X
X
Smith
D
D
Laursen
R A
RA
Andley
U P
UP
Reddan
J R
JR
Taylor
A
A
D000818:Animals; D002417:Cattle; D002474:Cell-Free System; D018387:Codon, Initiator / Q000502:physiology; D004847:Epithelial Cells / Q000201:enzymology*; D006801:Humans; D015151:Immunoblotting; D007527:Isoenzymes; D007908:Lens, Crystalline / Q000166:cytology* / Q000201:enzymology; D008025:Ligases / Q000502:physiology*; D051379:Mice; D011233:Precipitin Tests; D014176:Protein Biosynthesis; D012333:RNA, Messenger; D011817:Rabbits; D044764:Ubiquitin-Activating Enzymes; D044767:Ubiquitin-Protein Ligases
D018387:Codon, Initiator; D007527:Isoenzymes; D012333:RNA, Messenger; D044767:Ubiquitin-Protein Ligases; D008025:Ligases; D044764:Ubiquitin-Activating Enzymes