The unfolded protein response coordinates the production of endoplasmic reticulum protein and endoplasmic reticulum membrane.

The endoplasmic reticulum (ER) is a multifunctional organelle responsible for production of both lumenal and membrane components of secretory pathway compartments. Secretory proteins are folded, processed, and sorted in the ER lumen and lipid synthesis occurs on the ER membrane itself. In the yeast Saccharomyces cerevisiae, synthesis of ER components is highly regulated: the ER-resident proteins by the unfolded protein response and membrane lipid synthesis by the inositol response. We demonstrate that these two responses are intimately linked, forming different branches of the same pathway. Furthermore, we present evidence indicating that this coordinate regulation plays a role in ER biogenesis.     

Tail-anchored membrane protein insertion into the endoplasmic reticulum

Membrane proteins are inserted into the endoplasmic reticulum (ER) by two highly conserved parallel pathways. The well-studied co-translational pathway uses signal recognition particle (SRP) and its receptor for targeting and the SEC61 translocon for membrane integration. A recently discovered post-translational pathway uses an entirely different set of factors involving transmembrane domain (TMD)-selective cytosolic chaperones and an accompanying receptor at the ER. Elucidation of the structural and mechanistic basis of this post-translational membrane protein insertion pathway highlights general principles shared between the two pathways and key distinctions unique to each.     

Transmembrane domain-dependent partitioning of membrane proteins within the endoplasmic reticulum

The length and hydrophobicity of the transmembrane domain (TMD) play an important role in the sorting of membrane proteins within the secretory pathway; however, the relative contributions of protein-protein and protein-lipid interactions to this phenomenon are currently not understood. To investigate the mechanism of TMD-dependent sorting, we used the following two C tail-anchored fluorescent proteins (FPs), which differ only in TMD length: FP-17, which is anchored to the endoplasmic reticulum (ER) membrane by 17 uncharged residues, and FP-22, which is driven to the plasma membrane by its 22-residue-long TMD. Before export of FP-22, the two constructs, although freely diffusible, were seen to distribute differently between ER tubules and sheets. Analyses in temperature-blocked cells revealed that FP-17 is excluded from ER exit sites, whereas FP-22 is recruited to them, although it remains freely exchangeable with the surrounding reticulum. Thus, physicochemical features of the TMD influence sorting of membrane proteins both within the ER and at the ER-Golgi boundary by simple receptor-independent mechanisms based on partitioning.     

Photobleaching GFP reveals protein dynamics inside live cells

Cell biologists have used photobleaching to investigate the lateral mobility of fluorophores on the cell surface since the 1970s. Fusions of green fluorescent protein (GFP) to specific proteins extend photobleaching techniques to the investigation of protein dynamics within the cell, leading to renewed interest in photobleaching experiments. This article revisits general photobleaching concepts, reviews what can be learned from them and discusses applications illustrating the potential of photobleaching GFP fusion proteins inside living cells.     

Regulation of immature protein dynamics in the endoplasmic reticulum

The quality of nascent protein folding in vivo is influenced by the microdynamics of the proteins. Excessive collisions between proteins may lead to terminal misfolding, and the frequency of protein interactions with molecular chaperones determines their folding rates. However, it is unclear how immature protein dynamics are regulated. In this study, we analyzed the diffusion of immature tyrosinase in the endoplasmic reticulum (ER) of non-pigmented cells by taking advantage of the thermal sensitivity of the tyrosinase. The diffusion of tyrosinase tagged with yellow fluorescence protein (YFP) in living cells was directly measured using fluorescent correlation spectroscopy. The diffusion of folded tyrosinase in the ER of cells treated with brefeldin A, as measured by fluorescent correlation spectroscopy, was critically affected by the expression level of tyrosinase-YFP. Under defined conditions in which random diffusional motion of folded protein was allowed, we found that the millisecond-order diffusion rate observed for folded tyrosinase almost disappeared for the misfolded molecules synthesized at a nonpermissive high temperature. This was not because of enhanced aggregation at the high temperature, as terminally misfolded tyrosinase synthesized in the absence of calnexin interactions showed comparable, albeit slightly slower, diffusion. Yet, the thermally misfolded tyrosinase was not immobilized when measured by fluorescence recovery after photobleaching. In contrast, terminally misfolded tyrosinase synthesized in cells in which alpha-glucosidases were inhibited showed extensive immobilization. Hence, we suggest that the ER represses random fluctuations of immature tyrosinase molecules while preventing their immobilization.     

Regulation of protein biogenesis at the endoplasmic reticulum membrane

The biogenesis of most secretory and membrane proteins involves targeting the nascent protein to the endoplasmic reticulum (ER), translocation across or integration into the ER membrane and maturation into a functional product. The essential machinery that directs these events for model secretory and membrane proteins has been identified, shifting the focus of studies towards the molecular mechanisms by which these core components function. By contrast, regulatory mechanisms that allow certain proteins to serve multiple functions within a cell remain entirely unexplored. This article examines each stage of protein biogenesis as a potential site of regulation that could be exploited by the cell to effectively increase the diversity of functional gene expression.     

Posttranslational protein translocation across the membrane of the endoplasmic reticulum

Posttranslational protein translocation across the membrane of the endoplasmic reticulum is mediated by the Sec complex. This complex includes a transmembrane channel formed by multiple copies of the Sec61 protein. Translocation of a polypeptide begins when the signal sequence binds at a specific site within the channel. Binding results in the insertion of the substrate into the channel, possibly as a loop with a small segment exposed to the lumen. While bound, the signal sequence is in contact with both protein components of the channel and the lipid of the membrane. Subsequent movement of the polypeptide through the channel occurs when BiP molecules interact transiently with a luminal domain of the Sec complex, hydrolyze ATP, and bind to the substrate. Bound BiP promotes translocation by preventing the substrate from diffusing backwards through the channel, and thus acts as a molecular ratchet.     

Electron tomography reveals the endoplasmic reticulum as a membrane source for autophagosome formation

The origin and source of autophagosomal membranes are long-standing questions. By electron microscopy, we show that the endoplasmic reticulum (ER) associates with early autophagic structures called isolation membranes (IM) or phagophores in mammalian culture cells. Overexpression of a mutant of Atg4B, which causes defects in autophagosome formation, caused accumulation of ER-IM complexes. Electron tomography revealed the ER-IM complex as a subdomain of the ER forming a cradle encircling the IM, and showed that both ER and isolation membranes are interconnected.     

Inhibition of protein translocation across the endoplasmic reticulum membrane by sterols

Cholesterol and related sterols are known to modulate the physical properties of biological membranes and can affect the activities of membrane-bound protein complexes. Here, we report that an early step in protein translocation across the endoplasmic reticulum (ER) membrane is reversibly inhibited by cholesterol levels significantly lower than those found in the plasma membrane. By UV-induced chemical cross-linking we further show that high cholesterol levels prevent cross-linking between ribosome-nascent chain complexes and components of the Sec61 translocon, but have no effect on cross-linking to the signal recognition particle. The inhibiting effect on translocation is different between different sterols. Our data suggest that the protein translocation machinery may be sensitive to changes in cholesterol levels in the ER membrane.     

An ATP-binding membrane protein is required for protein translocation across the endoplasmic reticulum membrane.

The role of nucleotides in providing energy for polypeptide transfer across the endoplasmic reticulum (ER) membrane is still unknown. To address this question, we treated ER-derived mammalian microsomal vesicles with a photoactivatable analogue of ATP, 8-N3ATP. This treatment resulted in a progressive inhibition of translocation activity. Approximately 20 microsomal membrane proteins were labeled by [alpha 32P]8-N3ATP. Two of these were identified as proteins with putative roles in translocation, alpha signal sequence receptor (SSR), the 35-kDa subunit of the signal sequence receptor complex, and ER-p180, a putative ribosome receptor. We found that there was a positive correlation between inactivation of translocation activity and photolabeling of alpha SSR. In contrast, our data demonstrate that the ATP-binding domain of ER-p180 is dispensable for translocation activity and does not contribute to the observed 8-N3ATP sensitivity of the microsomal vesicles.     

Pleiotropic effects of membrane cholesterol upon translocation of protein across the endoplasmic reticulum membrane

Various proteins are translocated through and inserted into the endoplasmic reticulum membrane via translocon channels. The hydrophobic segments of signal sequences initiate translocation, and those on translocating polypeptides interrupt translocation to be inserted into the membrane. Positive charges suppress translocation to regulate the orientation of the signal sequences. Here, we investigated the effect of membrane cholesterol on the translocational behavior of nascent chains in a cell-free system. We found that the three distinct translocation processes were sensitive to membrane cholesterol. Cholesterol inhibited the initiation of translocation by the signal sequence, and the extent of inhibition depended on the signal sequence. Even when initiation was not inhibited, cholesterol impeded the movement of the positively charged residues of the translocating polypeptide chain. In surprising contrast, cholesterol enhanced the translocation of hydrophobic sequences through the translocon. On the basis of these findings, we propose that membrane cholesterol greatly affects partitioning of hydrophobic segments into the membrane and impedes the movement of positive charges.     

The protein translocation channel binds proteasomes to the endoplasmic reticulum membrane

Misfolded secretory proteins are transported across the endoplasmic reticulum (ER) membrane into the cytosol for degradation by proteasomes. A large fraction of proteasomes in a cell is associated with the ER membrane. We show here that binding of proteasomes to ER membranes is salt sensitive, ATP dependent, and mediated by the 19S regulatory particle. The base of the 19S particle, which contains six AAA-ATPases, binds to microsomal membranes with high affinity, whereas the 19S lid complex binds weakly. We demonstrate that ribosomes and proteasomes compete for binding to the ER membrane and have similar affinities for their receptor. Ribosomes bind to the protein conducting channel formed by the Sec61 complex in the ER membrane. We co-precipitated subunits of the Sec61 complex with ER-associated proteasome 19S particles, and found that proteoliposomes containing only the Sec61 complex retained proteasome binding activity. Collectively, our data suggest that the Sec61 channel is a principal proteasome receptor in the ER membrane.     

Microdomains of endoplasmic reticulum within the sarcoplasmic reticulum of skeletal myofibers

The relationship between the endoplasmic reticulum (ER) and the sarcoplasmic reticulum (SR) of skeletal muscle cells has remained obscure. In this study, we found that ER- and SR-specific membrane proteins exhibited diverse solubility properties when extracted with mild detergents. Accordingly, the major SR-specific protein Ca(2+)-ATPase (SERCA) remained insoluble in Brij 58 and floated in sucrose gradients while typical ER proteins were partially or fully soluble. Sphingomyelinase treatment rendered SERCA soluble in Brij 58. Immunofluorescence staining for resident ER proteins revealed dispersed dots over I bands contrasting the continuous staining pattern of SERCA. Infection of isolated myofibers with enveloped viruses indicated that interfibrillar protein synthesis occurred. Furthermore, we found that GFP-tagged Dad1, able to incorporate into the oligosaccharyltransferase complex, showed the dot-like structures but the fusion protein was also present in membranes over the Z lines. This behaviour mimics that of cargo proteins that accumulated over the Z lines when blocked in the ER. Taken together, the results suggest that resident ER proteins comprised Brij 58-soluble microdomains within the insoluble SR membrane. After synthesis and folding in the ER-microdomains, cargo proteins and non-incorporated GFP-Dad1 diffused into the Z line-flanking compartment which likely represents the ER exit sites.     

Degradation of proteins within the endoplasmic reticulum.

Certain newly synthesized proteins within the endoplasmic reticulum undergo rapid turnover by a non-lysosomal proteolytic pathway. Biochemical and morphological evidence has suggested that these proteins never leave the endoplasmic reticulum before they are degraded. The mechanism(s) for the selective targeting of proteins for degradation within the endoplasmic reticulum is still not understood, but appears to rely on specific structural determinants on the protein substrates. Important cellular functions are likely to be served by this endoplasmic reticulum degradative system, including disposal of abnormal proteins and the selective turnover of metabolically regulated proteins.     

Protein transport across the endoplasmic reticulum membrane

A decisive step in the biosynthesis of many eukaryotic proteins is their partial or complete translocation across the endoplasmic reticulum membrane. A similar process occurs in prokaryotes, except that proteins are transported across or are integrated into the plasma membrane. In both cases, translocation occurs through a protein-conducting channel that is formed from a conserved, heterotrimeric membrane protein complex, the Sec61 or SecY complex. Structural and biochemical data suggest mechanisms that enable the channel to function with different partners, to open across the membrane and to release laterally hydrophobic segments of membrane proteins into lipid.     

Segregation of the signal sequence receptor protein in the rough endoplasmic reticulum membrane

The signal sequence receptor (SSR), an integral membrane glycoprotein of 34 kDa, has previously been shown to be a component of the molecular environment which nascent polypeptide chains meet in passage through the endoplasmic reticulum (ER) membrane. We have used antibodies directed against the SSR and both immunocytochemistry and cell fractionation to determine its distribution in rat liver cells. SSR was found largely restricted to the rough ER. Only small amounts of the protein were detected in smooth ER. These results provide further evidence for a functional differentiation of rough and smooth ER and for a role of SSR in protein translocation across the ER membrane.     

Static retention of the lumenal monotopic membrane protein torsinA in the endoplasmic reticulum

TorsinA is a membrane-associated enzyme in the endoplasmic reticulum (ER) lumen that is mutated in DYT1 dystonia. How it remains in the ER has been unclear. We report that a hydrophobic N-terminal domain (NTD) directs static retention of torsinA within the ER by excluding it from ER exit sites, as has been previously reported for short transmembrane domains (TMDs). We show that despite the NTD's physicochemical similarity to TMDs, it does not traverse the membrane, defining torsinA as a lumenal monotopic membrane protein and requiring a new paradigm to explain retention. ER retention and membrane association are perturbed by a subset of nonconservative mutations to the NTD, suggesting that a helical structure with defined orientation in the membrane is required. TorsinA preferentially enriches in ER sheets, as might be expected for a lumenal monotopic membrane protein. We propose that the principle of membrane-based protein sorting extends to monotopic membrane proteins, and identify other proteins including the monotopic lumenal enzyme cyclooxygenase 1 (prostaglandin H synthase 1) that share this mechanism of retention with torsinA.