In eukaryotic cells, most proteins destined for membrane insertion or secretion are first processed in the endoplasmic reticulum (ER). Nascent polypeptide chains, synthesized by cytoplasmic ribosomes, enter the ER lumen at specialized sites in the ER membrane called translocons, which are complexes of several ER membrane proteins that associate to form a pore (Schnell and Herbert, 2003
). Sec61α, Sec61β and Sec61γ form the pore, and this trimeric complex is associated with other proteins including ERj1, Sec62 and Sec63 in mammals (Meyer et al., 2000
; Zimmermann et al., 2006
). Mutations in SEC63
cause polycystic liver disease (PCLD) in humans, a progressive disorder characterized by the presence of many (>20) cysts throughout the liver (Davila et al., 2004
; Everson et al., 2004
). PCLD often co-occurs in patients with autosomal dominant polycystic kidney disease (PCKD), but can also exist as a separate disease without kidney cysts (Torres et al., 2007
). Polycystic livers can grow up to ten times their normal size, resulting in significant patient morbidity. Although a few therapeutic interventions are available to slow cyst growth, only liver transplantation can change the course of the disease (Drenth et al., 2010
). It remains unclear how mutations in SEC63
cause liver cysts, but possibilities include disrupted trafficking of vital proteins such as polycystin-1, an integral cilia membrane protein mutated in PCKD (Fedeles et al., 2011
) and disrupted tethering of proteins to the cytosolic face of the ER (Müller et al., 2010
). Another possibility is that disruption of SEC63
triggers ER stress that contributes to the pathophysiology of PCLD.
Nascent polypeptides are transported across the ER translocon for processing, folding and maturation (Rapoport, 2007
). An imbalance between the load of unfolded preproteins that enter the ER and the capacity of this organelle to properly process the load results in ER ‘stress’: in this case an accumulation of misfolded proteins in the ER lumen (Ron and Walter, 2007
). This activates the unfolded protein response (UPR), a conserved cellular homeostatic mechanism, in an attempt to reconcile the imbalance. If the imbalance persists, the UPR can ultimately lead to cell death (Ron and Walter, 2007
). Not surprisingly, elevation of ER stress and activation of the UPR are implicated in the pathology of many diseases, including myelin disorders such as multiple sclerosis and Charcot-Marie-Tooth disease (D’Antonio et al., 2009
; Lin and Popko, 2009
Myelin is a multilayered membrane formed by the wrapping of glial cells around axons that allows for efficient conduction of action potentials in the vertebrate nervous system (Nave and Trapp, 2008
). Specialized glial cells generate the myelin sheath: oligodendrocytes in the central nervous system (CNS) and Schwann cells in the peripheral nervous system (PNS). Myelin is formed as an elaboration of the plasma membrane of the glial cells, which must generate tremendous amounts of membrane proteins and lipids (Anitei and Pfeiffer, 2006
). Segments of myelin are separated by the nodes of Ranvier, which are unmyelinated regions of axonal membrane containing a high density of voltage-gated sodium channels (NaV) (Salzer, 2003
; Salzer et al., 2008
). These channels propagate the action potential by generating current in response to membrane depolarization (Ritchie, 1995
Mutations in human SEC63 cause polycystic liver disease (PCLD). There are few treatment options for PCLD; only invasive surgery or liver transplantation can change the course of the disease. Although it is known that SEC63 is part of the endoplasmic reticulum (ER) translocon complex, which transports nascent polypeptides across membranes for folding and maturation in the ER, how mutations in SEC63 cause PCLD has been unclear. Among many hypotheses, it has been proposed that SEC63 mutations might trigger ER stress, which occurs when the capacity of the ER to process nascent or damaged proteins is overloaded. Many studies have linked ER stress with myelin disorders, including multiple sclerosis and peripheral neuropathy.
In a forward genetic screen for zebrafish mutants with abnormal sodium channel clustering in myelinated axons, the authors identified sec63st67, a missense mutation in sec63. Mutant larvae showed multiple defects in myelinated axons in the peripheral nervous system (PNS) and central nervous system (CNS), including disruptions in myelination. Additionally, liver development was abnormal in the sec63st67 mutants; however, cysts were not observed in the liver or kidneys. Swelling and fragmentation of the ER was observed in cells of the PNS, CNS and liver, and multiple molecular markers of ER stress were activated in sec63st67 mutants.
Implications and future directions
These findings introduce the sec63st67 mutant as a new model for studying the function of a gene implicated in PCLD, as well as the role of ER stress in disorders of disrupted myelination. Given that myelinating glia and hepatocytes synthesize large amounts of membrane and secreted proteins during development, they are highly susceptible to disruptions in the translocon machinery. This report advances our understanding of how ER stress contributes to disease processes and provides a new model for investigating the underlying mechanisms.
To discover genes required for the development and organization of myelinated axons, we performed a genetic screen in zebrafish to identify mutants with disruptions in the node of Ranvier (Voas et al., 2007
; Voas et al., 2009
) (M.G.V. and W.S.T., unpublished data). One mutation identified in this screen disrupts the zebrafish ortholog of the translocon protein Sec63. We show that the UPR is active in zebrafish sec63
mutants and that sec63
mutant axons in the CNS and the PNS are hypomyelinated, with reduced and abnormal NaV clusters. Given the role of Sec63 in human PCLD, we also examined the livers of zebrafish sec63
mutants. We show that pathology develops in this organ, with unusual accumulations of enlarged ER cisternae, disrupted bile canaliculi and accumulation of large, debris-laden lysosomes. These results raise the possibility that ER stress contributes to PCLD caused by SEC63
mutations and offer a new model for diseases involving protein trafficking and ER stress.