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REGULATION OF SECRETORY AND
MEMBRANE PROTEIN BIOGENESIS
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Ramanujan S. Hegde, MD, PhD, Head, Unit on Protein Biogenesis Sang-Wook
Kang, PhD, Postdoctoral Fellow Neena
Rane, PhD, Postdoctoral Fellow Ajay
Sharma, PhD, Research Fellow Corinna
Levine, BS, Postbaccalaureate Fellowa Niyathi
Hegde, Summer Student* Carolyn Eagan, Summer Student* |
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We study the mechanisms regulating
the synthesis, translocation, and maturation of secretory and membrane
proteins at the mammalian endoplasmic reticulum (ER). The translocon, a
complex macromolecular assembly at the ER, serves as a protein-conducting
channel through which substrates enter the secretory pathway. The translocon
participates in diverse cellular activities that range from the import of
secretory proteins, to topogenesis and assembly of complex multispanning
membrane proteins, to the export of misfolded substrates from the ER into the
cytosol for degradation. The mechanisms that allow this shared translocon to
accommodate such an extensive range of substrates both efficiently and
accurately remain largely unknown. The principal goal of our ongoing studies
is to define the molecular mechanisms and components of the translocon that
recognize the information in the primary sequence of its substrates to
mediate their proper vectorial transport, asymmetric topogenesis, and
membrane integration. By delineating the steps during the biosynthesis of
normal versus disease-associated variants of secretory and membrane proteins,
we are formulating hypotheses regarding the molecular basis of particular
diseases of the early secretory pathway and then testing them in vivo. Molecular mechanisms of
prion protein topogenesis Rane, Hegde The prion protein (PrP), a
brain glycoprotein involved in various neurodegenerative diseases, has proven
to be a particularly instructive example of complex and highly regulated
translocation. In addition to its notoriety as the putative “protein-only”
infectious agent in prion diseases, the biogenesis of PrP at the ER is
unusual in that an initially homogeneous cohort of nascent PrP chains gives
rise to three distinct topologic forms: a fully translocated form (termed secPrP)
and two transmembrane forms that span the membrane in opposite orientations (NtmPrP
and CtmPrP). In vivo
studies have revealed that even a slight overrepresentation of the CtmPrP
topologic form results in the development of neurodegenerative disease in
both mouse model systems and naturally occurring human disease. Our current
efforts focus on dissecting the mechanisms that direct an initially
homogeneous cohort of nascent PrP polypeptides into multiple topologic forms.
We recently discovered that segregation of nascent PrP into different
topologic forms is critically dependent on the precise timing of signal
sequence–mediated initiation of N-terminus translocation. Consequently, this
step could be experimentally tuned to modify PrP topogenesis, including
complete reversal of the elevated CtmPrP caused by
disease-associated mutations in the transmembrane domain. We have created
transgenic mice to determine whether CtmPrP-mediated
neurodegeneration can be averted by modulating this newly discovered step
during PrP biogenesis. Parallel biochemical studies
employing the solubilization, fractionation, and reconstitution of ER
membrane proteins have demonstrated that regulatory trans-acting factors are absolutely required for PrP to be
synthesized in the proper ratio of its topologic forms. We have now purified
two of these factors and have identified them as the translocon-associated
protein complex (TRAP) and protein disulfide isomerase (PDI). Analysis of PrP
translocation intermediates suggests that TRAP and PDI act sequentially to
facilitate translocation of PrP’s N-terminus into the ER lumen, the decisive
event in determining its topology. Ongoing studies are investigating the role
of these newly discovered factors in the biogenesis of other substrates and
their potential role in the pathogenesis of PrP-associated neurodegeneration. A
principal strategy for controlling the progression of various
neurodegenerative diseases is to reduce the total cellular burden of the
potentially cytotoxic forms of the responsible protein. In diseases
associated with the PrP, neurodegeneration may be mediated by several variant
isoforms that include a transmembrane form (termed CtmPrP; see
above) and a more recently described cytoplasmic form. To gain insight into
how these variants are initially generated, we have dissected the pathway of
PrP biogenesis at the ER (see above). Our results indicate that the decisive
event in avoiding the generation of both these forms is the signal sequence–mediated
translocation of the N-terminus of PrP into the ER lumen. This critical step
in PrP translocation is modulated by TRAP, a heterotetrameric membrane
protein of previously unknown function, and ER lumenal chaperones. By using a
constitutive, highly efficient signal sequence that does not depend on these
factors for efficient N-terminus translocation, the generation of CtmPrP
and cytosolic PrP can be substantially reduced. The consequences of this
manipulation in a cultured neuronal cell line are a marked reduction of cytotoxic
and aggregation-prone variants of PrP and protection from apoptotic cell
death. The results define a pathway for the normal biogenesis of PrP and
demonstrate that the total cellular burden of cytotoxic forms of PrP is
controlled primarily during its initial translocation into the ER. Some of
our ongoing studies focus on examining the consequences in transgenic mice of
altering the cellular burden of cytoplasmic forms of PrP. In particular, we
wish to determine whether reducing the amounts of this potentially neurotoxic
species of PrP confers protection against neurodegeneration during the course
of transmissible prion disease. Hegde
RS, Kim
SJ, Hegde RS. Cotranslational partitioning of nascent prion protein into multiple
populations at the translocation channel. Mol
Biol Cell 2002;13:3775-3786. Mechanisms of calreticulin
multifunctionality Sharma, Hegde The generation of multiple
topologic forms of the PrP has raised the provocative possibility that a
single protein can exist in several spatially and functionally discrete
populations. We have therefore sought to identify and characterize examples
of proteins whose translocation into the ER is productively regulated to
generate multiple functional forms. To this end, we have been studying the
protein calreticulin, an abundant calcium-binding chaperone of the ER lumen.
Remarkably, calreticulin has also been implicated in functions outside the
ER, such as nuclear export, binding to and modulating activity of steroid
hormone receptors, and binding to and influencing integrin signaling. How
this multifunctional protein might perform these functions in several
cellular compartments remains unknown. We recently discovered that the signal
sequence of calreticulin is naturally designed to allow a small but discrete
population of the protein to be retained in the cytoplasm. The cytoplasmic
population can be reduced by using a signal sequence from another protein,
prolactin. Thus, we have identified a potential mechanism by which a protein
can be made in more than one population as well as a means to manipulate such
heterogeneity. Using both cell culture and transgenic mouse models, we are
now examining the relative contributions of the different populations of
calreticulin to each of its several functions. We hope to illuminate general
principles by which cells can regulate protein localization, and hence
function, to meet changing cellular demands with stress, cell cycle,
differentiation, or disease pathogenesis. Regulation of protein
biogenesis by signal sequences Levine, Our discovery that the
N-terminal signal sequence of PrP regulates its topology established a new
function for this domain independent of its well-studied role in protein targeting.
Using signal sequence mutants that uncouple the domain’s targeting and
post-targeting functions, we demonstrated that the post-targeting functions
in gating the translocation channel to provide the nascent polypeptide access
to the ER lumen. We subsequently developed a novel assay for signal-mediated
translocon gating to demonstrate that signal sequences display a remarkable
degree of variation in initiating nascent chain access to the lumenal
environment. We found that such substrate-specific properties of signals were
evolutionarily conserved, functionally matched to their respective mature
domains, and important for the proper biogenesis of some proteins. A recent
analysis of several naturally occurring disease-associated mutants in signal
sequences revealed that many mutants are altered in their gating but not, as
previously assumed, in their targeting function. Thus, we discovered that the
long-observed sequence variations of signals do not simply represent
functional degeneracy but instead encode differences in translocon gating
that are critical to the proper biogenesis of some secretory and membrane
proteins. We are currently investigating whether the cell has exploited the
substrate-specific properties of signal sequences to regulate the subcellular
localization of certain proteins, such as calreticulin, that have been shown
to be present in several compartments. Hegde
RS. Targeting and beyond: new roles for old signal sequences. Mol Cell 2002;10:697-698. Kim
SJ, Mitra D, Levine
CG, Mitra D, Sharma A, Smith C, Hegde RS. The efficiency of protein
compartmentalization into the secretory pathway. Mol Biol Cell 2004, in
press. Lingappa
VR, Rutkowski DT, Hegde RS, Andersen OS. Conformational control through
translocational regulation: a new view of secretory and membrane protein
folding. Bioessays 2002;24:741-748. Structural and
functional analysis of the TRAP complex Hegde; in
collaboration with Akey, Lippincott-Schwartz, Snapp The search for factors involved
in PrP topogenesis led to our purification of the TRAP complex, a set of four
proteins with a previously unknown function. Our recent studies established
that TRAP is required for the translocation of only some substrates, a
substrate specificity that is encoded in the signal sequence. Remarkably,
TRAP specifically aids vectorial transport of substrates whose signal
sequences, after mediating targeting to the ER, are delayed in their gating
of the translocon. Thus, it appears that TRAP is a critical component of the
translocation machinery that aids in decoding the substrate-specific
information in signal sequences. Our current studies of TRAP function follow
two approaches and focus on understanding the molecular mechanism by which it
facilitates signal recognition, substrate translocation, and protein
topogenesis. In collaboration with Christopher Akey’s group, we are using
cryo-electron microscopy to compare the structures of ribosome-translocon
complexes that have been prepared with and without the TRAP complex. The
studies are providing the initial structural views of not only the TRAP
complex but also of its relative position within the translocon. In parallel,
we are investigating the consequence in
vivo of RNAi-mediated suppression of TRAP expression in cultured cells.
We expect that the structural information combined with the functional
analyses will provide insight into the mechanisms by which TRAP can regulate
protein translocation in a substrate-specific manner. Small molecule
inhibitors of protein translocation Rane, Hegde; in
collaboration with Garrison, To develop new methods and
probes for protein translocation, we have been developing pharmacologic
methods of modulating translocation in
vivo. We have recently synthesized and characterized a novel small
molecule inhibitor of co-translational protein translocation. We demonstrated
the compound both in vitro and in
cultured cells and showed that it inhibits the translocation of only some
proteins. Given that the substrate specificity is encoded in the signal
sequence, the sensitivity or resistance to the inhibitor can be conferred to
any protein of interest simply by choice of signal. We have identified the
target of this inhibitor, which appears to be the Sec61 complex, the central
component of the protein translocation channel. These tools and findings open
the way for selectively and potently modulating the translocation of
individual substrates in live cells, an approach that we are applying to
investigating the role of protein translocation in various cellular events,
such as protein aggregation and toxicity, protein degradation, and the
cellular response to ER stress. Visualization of
translocon organization in cells Hegde; in
collaboration with Lippincott-Schwartz, Snapp A qualitatively different facet
of protein biogenesis is the question of where
within the ER various events occur. The translocon participates in diverse
cellular activities that range from the import of secretory proteins, to
topogenesis and assembly of complex multispanning membrane proteins, to the
export of misfolded substrates from the ER to the cytosol for degradation. We
wished to determine whether all these translocon-associated activities are
distributed homogeneously throughout the ER or are organized and regulated in
spatial and temporal dimensions to meet the changing needs of the cell. At
present, we have little or no insight into this question, largely because the
current approaches to understanding protein translocation use biochemical
systems in which spatial relationships are lost. In collaboration with the
laboratory of Jennifer Lippincott-Schwartz, we are using biophysical
techniques such as fluorescence resonance energy transfer (FRET) to probe in situ the molecular organization of
the components of the translocation machinery. In initial studies, we used
analysis of FRET between subunits of the Sec61p complex, a principal
component of the ER protein translocon, to monitor directly the assembly
state of the translocon in cells. Our studies revealed that, while the
translocon can be assembled from its components in response to ligands for
protein translocation in biochemical systems, it does not disassemble and
reassemble between successive rounds of transport in vivo. Instead, an actively engaged translocon is distinguished
from a quiescent translocon by conformational changes that can be directly
detected by differences in FRET. By correlating the formation of particular
protein complexes with biochemical activities, we endeavor to visualize
directly the functional segregation and organization of the ER and to monitor
potential changes in it during cellular metabolism, development, or disease
pathogenesis. Snapp EL, COLLABORATORS Christopher Akey,
PhD, Jennifer Garrison,
BS, Jennifer
Lippincott-Schwartz, PhD, Cell Biology
and Metabolism Branch, NICHD, Erik Snapp, PhD, Cell Biology and Metabolism Branch, NICHD,
Jack Taunton, PhD, aLeft NICHD in August 2004. For
further information, contact hegder@mail.nih.gov |
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