Functions of the Hsp90-Binding FKBP Immunophilins - The Networking of Chaperones by Co-chaperones - page 49

that the Hsp90-binding immunophilin FKBPL/WISp39 also favors GR retrotrans-
port in a similar fashion as FKBP52 (McKeen et al. 2008 ).
Importantly, the active Hsp90-, FKBP52-dependent mechanism for cytoplasmic
transport first described for GR has also been found for other factors such as MR
(Galigniana et al. 2010b ), AR (Thomas et al. 2006 ), ecdysone receptor (Vafopou-
lou and Steel 2012 ), p53 (Galigniana et al. 2004b ), RAC3 (Colo et al. 2008 ), and
adeno-associated virus-2 (AAV) (Zhao et al. 2006 ). This immunophilin-dependent
model for soluble protein trafficking implies that the proteins of the heterocomplex
should remain associated to the client cargo during the passage through the nuclear
pore complex. In line with this speculation, it was demonstrated that the whole
Hsp90-FKBP52 heterocomplex cross-linked to corticosteroid receptors (Galigni-
ana et al. 2010b ; Echeverria et al. 2009 ) is able to translocate intact in a hormone-
dependent manner through the nuclear pore of digitonin-permeabilized cells, sug-
gesting that steroid-receptor transformation and its subsequent dimerization must
be a nuclear event. This was recently confirmed by using different methodologies
(Galigniana et al. 2010b ; Grossmann et al. 2012 ; Presman et al. 2014 ; Presman
et al. 2010 ).
Studies of reconstitution of the Hsp90-FKBP52 heterocomplex with purified
proteins or reticulocyte lysate as a source of chaperones (Echeverria et al. 2009 ),
demonstrated that the interaction of GR with structures of the nuclear pore such
as nucleoporins (NUPs) is strengthened when both factors, GR and NUPs, are
chaperoned. On the other hand, the discovery that NUPs are Hsp90- and FKBP52-
interacting proteins also suggests a potential regulatory role of these chaperones
for the nuclear import process. In this regard, it has always been very difficult to
explain how single factors such as importins could shield the multitude of differ-
ent protein-, RNA- and DNA-binding domains in transport cargoes that are import
substrates. It could be speculated that these chaperones associated to importins,
NUPs, and the cargo itself may act as a cooperative system to prevent aggregation
of cargoes when a hydrophobic domain is exposed during the translocation step.
This may justify why there is a more efficient interaction between NUPs and GR
when both proteins are associated to the Hsp90-FKBP52 complex compared to
both 'naked' proteins (Echeverria et al. 2009 ).
The association of FKBP52 and PP5 with Nup62 seems to be Hsp90-depen-
dent, as was suggested by the almost-complete dissociation of these immunophil-
ins from Nup62 in the presence of the Hsp90-disrupting agent radicicol (Echever-
ria et al. 2009 ). However, indirect immunofluorescence assays performed with
intact cells treated with radicicol still show the presence of the immunophilins in
the perinuclear ring, suggesting that they may also bind in an Hsp90-independent
manner to other perinuclear structures. Nonetheless, competition experiments
with the TPR domain overexpressed in intact cells showed that the perinuclear
signal of FKBP52 was totally abolished, indicating that most, if not all, types
of association of the immunophilin with any structure of the nuclear envelope
require the TPR domain.