Kinesin Adapter JLP Links PIKfyve to Microtubule-based
Endosome-to-Trans-Golgi Network Traffic of
Furin |
| |
Authors: | Ognian C Ikonomov Jason Fligger Diego Sbrissa Rajeswari Dondapati Krzysztof Mlak Robert Deeb and Assia Shisheva |
| |
Institution: | Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan 48201 |
| |
Abstract: | JIPs (c-Jun N-terminal kinase interacting
proteins), which scaffold JNK/p38 MAP kinase signaling modules,
also bind conventional kinesins and are implicated in microtubule-based
membrane trafficking in neuronal cells. Here we have identified a novel splice
variant of the Jip4 gene product JLPL (JNK-interacting
leucine zipper protein) in yeast-two hybrid screens with the phosphoinositide
kinase PIKfyve. The interaction was confirmed by pulldown and
coimmunoprecipitation assays in native cells. It engages the PIKfyve
cpn60_TCP1 consensus sequence and the last 75 residues of the JLP C terminus.
Subpopulations of both proteins cofractionated and populated similar
structures at the cell perinuclear region. Because PIKfyve is essential in
endosome-to-trans-Golgi network (TGN) cargo transport, we tested whether JLP
is a PIKfyve functional partner in this trafficking pathway. Short interfering
RNA (siRNA)-mediated depletion of endogenous JLP or PIKfyve profoundly delayed
the microtubule-based transport of chimeric furin (Tac-furin) from endosomes
to the TGN in a CHO cell line, which was rescued upon ectopic expression of
siRNA-resistant JLP or PIKfyve constructs. Peptides from the contact sites in
PIKfyve and JLP, or a dominant-negative PIKfyve mutant introduced into cells
by ectopic expression or microinjection, induced a similar defect. Because
Tac-TGN38 delivery from endosomes to the TGN, unlike that of Tac-furin, does
not require intact microtubules, we monitored the effect of JLP and PIKfyve
depletion or the interacting peptides administration on Tac-TGN38 trafficking.
Remarkably, neither maneuver altered the Tac-TGN38 delivery to the TGN. Our
data indicate that JLP interacts with PIKfyve and that both proteins and their
association are required in microtubule-based, but not in
microtubule-independent, endosome-to-TGN cargo transport.In mammalian cells, the endosomal/endocytic system comprises an
interconnected and morphologically complex network of membrane organelles that
supports fundamental functions such as nutrient entry and delivery for
degradation, removal and degradation of plasma membrane or Golgi proteins,
regulation and integration of signaling pathways, and protein recycling to the
cell surface or the
TGN2
(1–4).
From the plasma membrane, the endocytosed cargo is first delivered to early
endosomes/sorting endosomes. Cargoes destined for recycling to the cell
surface then enter the endocytic recycling compartment, whereas others,
intended for degradation, remain in early endosomes. Early endosomes undergo a
series of changes, known as maturation, to give rise to maturing transport
intermediates (herein ECV/MVBs; also Ref.
5) and to late endosomes that
fuse with lysosomes to deliver cargo for degradation. Recycling or degradation
is not the only outcome of the cell surface-originated cargoes. A set of
internalized transmembrane proteins, including intracellular sorting
receptors, enzymes, and toxins, are retrieved from the endosomal system and
transported to the TGN. The endosome-to-TGN trafficking of the
acid-hydrolase-sorting receptor, CI-MPR, the endopeptidase furin, and the
putative cargo receptor TGN38 are the best studied examples. These cargoes are
highly enriched in the TGN at steady state but arrive there from different
compartments, utilizing distinct mechanisms. Thus, TGN38 enters the TGN from
the endocytic recycling compartment by an iterative removal from the latter
compartment, furin reaches the TGN by exiting the early/late endosomal system,
and CI-MPR implements features of both pathways
(4,
6–9).Whereas the detailed molecular and cellular mechanisms underlying the
membrane progression in the course of cargo transport through the endosomal
system or retrieval from early/late endosomes to the TGN is still elusive,
experimental evidence has been accumulating to implicate PIKfyve, the sole
enzyme for PtdIns(3,5)P2 synthesis
(10). Thus, PIKfyve has been
found to interact with the late endosome-to-TGN transport factor Rab9 effector
p40 (11). Furthermore,
disruption of the PtdIns(3,5)P2 homeostatic mechanism by means of
expression of dominant-negative kinase-deficient point mutants of PIKfyve,
protein depletion, or pharmacological inhibition of PIKfyve activity was found
to impair the exit of a subset of cargoes from early endosomes to the TGN and
late endosomes or from the late endosomes
(12–16).
Phenotypically, these defects are manifested by progressive endosome swelling
and cytoplasmic vacuolation, first seen by expression of dominant-negative
PIKfyveK1831E in a number of mammalian cell types
(17) and confirmed thereafter
by other maneuvers inhibiting PIKfyve protein expression or activity
(14,
16). In vitro
reconstitution assays indicate that PIKfyve enzymatic activity is required in
endosome processing in two ways. It triggers the formation/fission (or
maturation) of ECV/MVBs from early endosomes and arrests the rate of fusion
events in the endosomal system
(18,
19). It is thus conceivable
that impaired PIKfyve and PtdIns(3,5)P2 functioning in the fission
and fusion events mechanistically underlies the constraints in the trafficking
pathways traversing endosomes.Microtubules aided by the microtubule-associated motor protein families of
kinesin and dynein are required for proper performance of the
endosomal/endocytic membrane system. Although their role is rather complex and
not completely understood, in vivo and in vitro studies
implicate microtubule-based dynamics in multiple aspects of the endocytic
trafficking, including sorting of endocytic contents, fission/fusion events at
early or late endosomes, early endosome maturation, and efficient motility of
the transport vesicles to their destination
(20–27).
Accumulating evidence indicates that the binding of motor proteins to
organelles or carrier vesicles is regulated by motor protein adapters.
Intriguingly, this newly emerging adapter function has been found to be
executed by proteins known as scaffolds of stress signaling enzymes. One such
adapter for conventional kinesins is the group of JIPs that scaffold the
JNK/p38 MAP kinase signaling modules
(28–31).
A mutation that causes mislocalization of synaptic vesicles and aberrant
axonal transport in Drosophila and Caenorhabditis elegans
affects the JIP3 homologs Sunday driver (dSYD) and Unc16,
respectively (32,
33). In mammalian cells, JIPs
are represented by four proteins (JIP1–4) derived from separate genes
and several alternatively spliced variants. JIP1, the founding member, is
structurally related to JIP2
(34,
35). JIP3 (also known as
Unc16/JSAP1/dSYD) is structurally unrelated to JIP1 or JIP2, but as those two,
it is abundant in neuronal cells
(30,
32,
36). The latest addition to
the group is JIP4 that occurs in three splice variants known thus far: JLP and
JIP4 in mouse and SPAG9 in humans
(31,
37,
38). JIP4, JLP, and SPAG9
(gene symbol, SPAG9) are structurally homologous to JIP3 but display
broader distribution
(37–39).
Remarkably, all four members of the JIP group interact with the kinesin1 light
chain, and potential cargoes for microtubule-based vesicle transport have been
proposed for JIP1–JIP3
(32,
33,
38,
40–43).
The role of JLP/JIP4 in the context of cargo transport or membrane trafficking
events, however, has never been investigated. In the present study we report
that JLP is a PIKfyve physical and functional partner in microtubule-based
endosome-to-TGN trafficking. The interaction is identified by a yeast
two-hybrid screen with the PIKfyve cpn60_TCP1 consensus sequence and mapped to
the 75-aa peptide fragment of the extreme JLP C terminus. By monitoring
divergent routes of cargo delivery to the TGN, differing by the requirement of
microtubule-dependent early endosome maturation, we have determined that JLP
assists PIKfyve selective functionality in microtubule-based endosome-to-TGN
trafficking. |
| |
Keywords: | |
|
|