Production of Cells with Targeted Integration of Gene Variants of Human ABC Transporter for Stable and Regulated Expression Using the Flp Recombinase System - Protein Misfolding and Cellular Stress in Disease and Aging - page 134

While the native conformation of a protein lies encoded in its
primary amino acid sequence, the ER greatly enhances protein
folding efficacy ( 2 ). During de novo synthesis in the ER, cysteine
disulfide bonds are formed and oligosaccharides are added to
asparagine ( N -linked glycosylation), serine/threonine residues
( O -linked glycosylation) of glycoproteins. The ER has a unique
oxidizing potential that supports disulfide bond formation
during protein folding ( 3 ). In addition to the well-known oxi-
doreductases, such as PDI and ERp57, many novel oxidoreductases
have been identified over the past few years whose functions and
substrates are unknown ( 4, 5 ). On the other hand, N -linked gly-
cans are added en blocc to proteins as “core oligosaccharides”
(Glc 3 Man 9 GlcNAc 2 ). Calnexin (CNX) is located near the translo-
con and can interact with nascent peptide chains of N -glycosylated
proteins. N -linked glycans are subjected to extensive modifica-
tion as glycoproteins mature and move through the ER via the
Golgi apparatus to their final destination, for example, the plasma
membrane.
Efficient quality control systems have evolved to prevent
incompletely folded molecules from moving along the secretory
pathway ( 1, 6-10 ). Accumulation of misfolded proteins in the ER
would detrimentally affect cellular functions. Misfolded proteins
are considered to be removed from the ER by retrotranslocation
to the cytosol compartment where they are then degraded by the
ubiquitin-proteasome system. This process is known as ER-associated
degradation (ERAD). At present, however, it remains unclear how
misfolded membrane proteins are selected and destroyed during
ERAD. Chaperone proteins are considered to solubilize aggrega-
tion-prone motifs. In the case of the yeast ATP-binding cassette
(ABC) transporter Ste6p, a 12-transmembrane protein, it has
recently been shown that Hsp70/40s act before ubiquitination
and facilitate the association of Ste6p with an E3 ubiquitin ligase
( 11 ). Furthermore, polyubiquitination was a prerequisite for
retrotranslocation, which required the Cdc48 complex and ATP
( 11 ). Thus, it is becoming increasingly important to identify and to
characterize multiple chaperone proteins that control the folding
and degradation of ABC transporter proteins.
A total of 48 human ABC transporter genes have been identi-
fied and sequenced ( 12 ). On the basis of the arrangement of mole-
cular structural components, that is, nucleotide binding domains
and topologies of transmembrane domains, these reported human
ABC transporters are classified into seven different sub-families
(A to G). Human ABC transporter ABCG2 (BCRP/MXR/ABCP)
belongs to the G subfamily of the ABC transporter proteins
( 13 ). This efflux pump is suggested to be responsible for pro-
tecting the body from toxic xenobiotics and for removing toxic
metabolites ( 14 ). Human ABCG2 is a so-called “half ABC trans-
porter” bearing a single ATP-binding fold at the NH 2 -terminus