Protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase

Protein synthesis and the folding of the newly synthesized proteins into the correct three-dimensional structure are coupled in cellular compartments of the exocytosis pathway by a process that modulates the phosphorylation level of eukaryotic initiation factor-2alpha (eIF2alpha) in response to a stress signal from the endoplasmic reticulum (ER). Activation of this process leads to reduced rates of initiation of protein translation during ER stress. Here we describe the cloning of perk, a gene encoding a type I transmembrane ER-resident protein. PERK has a lumenal domain that is similar to the ER-stress-sensing lumenal domain of the ER-resident kinase Ire1, and a cytoplasmic portion that contains a protein-kinase domain most similar to that of the known eIF2alpha kinases, PKR and HRI. ER stress increases PERK's protein-kinase activity and PERK phosphorylates eIF2alpha on serine residue 51, inhibiting translation of messenger RNA into protein. These properties implicate PERK in a signalling pathway that attenuates protein translation in response to ER stress.     

A di-acidic signal required for selective export from the endoplasmic reticulum

Transport of membrane proteins between intracellular compartments requires specific sequences in the protein cytoplasmic domain to direct packaging into vesicle shuttles. A sequence that mediates export from the endoplasmic reticulum (ER) has proved elusive. A di-acidic signal (Asp-X-Glu, where X represents any amino acid) on the cytoplasmic tail of vesicular stomatitis virus glycoprotein (VSV-G) and other cargo molecules was required for efficient recruitment to vesicles mediating export from the ER in baby hamster kidney cells. The existence of such a signal provides evidence that export from the ER occurs through a selective mechanism.     

Der3p/Hrd1p is required for endoplasmic reticulum-associated degradation of misfolded lumenal and integral membrane proteins

We have studied components of the endoplasmic reticulum (ER) proofreading and degradation system in the yeast Saccharomyces cerevisiae. Using a der3-1 mutant defective in the degradation of a mutated lumenal protein, carboxypeptidase yscY (CPY*), a gene was cloned which encodes a 64-kDa protein of the ER membrane. Der3p was found to be identical with Hrd1p, a protein identified to be necessary for degradation of HMG-CoA reductase. Der3p contains five putative transmembrane domains and a long hydrophilic C-terminal tail containing a RING-H2 finger domain which is oriented to the ER lumen. Deletion of DER3 leads to an accumulation of CPY* inside the ER due to a complete block of its degradation. In addition, a DER3 null mutant allele suppresses the temperature-dependent growth phenotype of a mutant carrying the sec61-2 allele. This is accompanied by the stabilization of the Sec61-2 mutant protein. In contrast, overproduction of Der3p is lethal in a sec61-2 strain at the permissive temperature of 25 degrees C. A mutant Der3p lacking 114 amino acids of the lumenal tail including the RING-H2 finger domain is unable to mediate degradation of CPY* and Sec61-2p. We propose that Der3p acts prior to retrograde transport of ER membrane and lumenal proteins to the cytoplasm where they are subject to degradation via the ubiquitin-proteasome system. Interestingly, in ubc6-ubc7 double mutants, CPY* accumulates in the ER, indicating the necessity of an intact cytoplasmic proteolysis machinery for retrograde transport of CPY*. Der3p might serve as a component programming the translocon for retrograde transport of ER proteins, or it might be involved in recognition through its lumenal RING-H2 motif of proteins of the ER that are destined for degradation.     

The membrane protein alkaline phosphatase is delivered to the vacuole by a route that is distinct from the VPS-dependent pathway

Membrane trafficking intermediates involved in the transport of proteins between the TGN and the lysosome-like vacuole in the yeast Saccharomyces cerevisiae can be accumulated in various vps mutants. Loss of function of Vps45p, an Sec1p-like protein required for the fusion of Golgi-derived transport vesicles with the prevacuolar/endosomal compartment (PVC), results in an accumulation of post-Golgi transport vesicles. Similarly, loss of VPS27 function results in an accumulation of the PVC since this gene is required for traffic out of this compartment. The vacuolar ATPase subunit Vph1p transits to the vacuole in the Golgi-derived transport vesicles, as defined by mutations in VPS45, and through the PVC, as defined by mutations in VPS27. In this study we demonstrate that, whereas VPS45 and VPS27 are required for the vacuolar delivery of several membrane proteins, the vacuolar membrane protein alkaline phosphatase (ALP) reaches its final destination without the function of these two genes. Using a series of ALP derivatives, we find that the information to specify the entry of ALP into this alternative pathway to the vacuole is contained within its cytosolic tail, in the 13 residues adjacent to the transmembrane domain, and loss of this sorting determinant results in a protein that follows the VPS-dependent pathway to the vacuole. Using a combination of immunofluorescence localization and pulse/chase immunoprecipitation analysis, we demonstrate that, in addition to ALP, the vacuolar syntaxin Vam3p also follows this VPS45/27-independent pathway to the vacuole. In addition, the function of Vam3p is required for membrane traffic along the VPS-independent pathway.     

Na,K-ATPase transport from endoplasmic reticulum to Golgi requires the Golgi spectrin-ankyrin G119 skeleton in Madin Darby canine kidney cells

Spectrin (betaISigma*) and ankyrin (AnkG119) associate with Golgi membranes and the dynactin complex, but their role in vesicle trafficking remains uncertain. We find that the actin-binding domain and membrane-association domain 1 (MAD1) of betaI spectrin together form a constitutive Golgi targeting signal in transfected MDCK cells. Expression of this signal in transfected cells disrupts the endogenous Golgi spectrin skeleton and blocks transport of alpha- and beta-Na,K-ATPase and vesicular stomatitis virus-G protein from the endoplasmic reticulum (ER) but does not disrupt the formation of Golgi stacks, the distribution of beta-COP, or the transport and surface display of E-cadherin. The Golgi spectrin skeleton is thus required for the transport of a subset of membrane proteins from the ER to the Golgi. We postulate that together with polyfunctional adapter proteins such as AnkG119, Golgi spectrin forms a docking complex that acts prior to the cis-Golgi, presumably with vesicular-tubular clusters (VTCs or ERGIC), to sequester specific membrane proteins into vesicles transiting between the ER and Golgi, and subsequently (probably involving other isoforms of spectrin and ankyrin) to mediate cargo transport within the Golgi and to other membrane compartments. We hypothesize that this vesicular spectrin-ankyrin adapter-protein trafficking (or tethering) system (SAATS) mediates the capture and transport of many membrane proteins and acts in conjunction with vesicle-targeting molecules to effect the efficient transport of cargo proteins.     

Topogenesis of a microsomal cytochrome P450 and induction of endoplasmic reticulum membrane proliferation in Saccharomyces cerevisiae

Cytochrome P450 52A3 (P450Cm1) is one of the membrane proteins known to trigger by its high-level expression a marked proliferation of the endoplasmic reticulum, (ER). To gain insight into the relationship between the expression of a membrane protein and the induction of ER proliferation we have characterized the membrane topology of P450Cm1 and identified the structural determinants required for ER targeting, formation of correct membrane orientation, and ER retention. We show that all these features are interrelated and determined by sequence elements within the NH2-terminal region of P450Cm1. Using several approaches--a protease protection assay followed by probing with peptide-specific antibodies, immunolabeling of the intact membrane-bound P450 protein, and expression of fusion proteins in Saccharomyces cerevisiae--membrane topology was defined as follows: residues 2-16 are located in the ER lumen, only the first hydrophobic segment (residues 17-34) spans the membrane, a second hydrophobic segment (48-66) is exposed at the cytoplasmic side, and the remaining part (67-523) forms a large cytosolic domain. Fused to a cytosolic reporter protein, the first 44-amino-acid sequence of P450Cm1 was sufficient to mediate ER targeting, wild-type membrane orientation, and retention in the ER. Similar to wild-type P450Cm1, various fusion proteins were able to induce distinctly organized structures of proliferated ER provided that they were either permanently retained in the ER or accumulated in this compartment due to a delay in further transportation. Thus, we conclude that membrane insertion of the first hydrophobic segment is sufficient to deliver a signal for increased membrane formation.     

A novel direct interaction of endoplasmic reticulum with microtubules

The positioning and dynamics of organelles in eukaryotic cells critically depend on membrane-cytoskeleton interactions. Motor proteins play an important role in the directed movement of organelle membranes along microtubules, but the basic mechanism by which membranes stably interact with the microtubule cytoskeleton is largely unknown. Here we report that p63, an integral membrane protein of the reticular subdomain of the rough endoplasmic reticulum (ER), binds microtubules in vivo and in vitro. Overexpression of p63 in cell culture led to a striking rearrangement of the ER and to concomitant bundling of microtubules along the altered ER. Mutational analysis of the cytoplasmic domain of p63 revealed two determinants responsible for these changes: an ER rearrangement determinant near the N-terminus and a central microtubule-binding region. The two determinants function independently of one another as indicated by deletion experiments. A peptide corresponding to the cytoplasmic tail of p63 promoted microtubule polymerization in vitro. p63 is the first identified integral membrane protein that can link a membrane organelle directly to microtubules. By doing so, it may contribute to the positioning of the ER along microtubules.     

The protein kinase Pak3 positively regulates Raf-1 activity through phosphorylation of serine 338

The pathway involving the signalling protein p21Ras propagates a range of extracellular signals from receptors on the cell membrane to the cytoplasm and nucleus. The Ras proteins regulate many effectors, including members of the Raf family of protein kinases. Ras-dependent activation of Raf-1 at the plasma membrane involves phosphorylation events, protein-protein interactions and structural changes. Phosphorylation of serine residues 338 or 339 in the catalytic domain of Raf-1 regulates its activation in response to Ras, Src and epidermal growth factor. Here we show that the p21-activated protein kinase Pak3 phosphorylates Raf-1 on serine 338 in vitro and in vivo. The p21-activated protein kinases are regulated by the Rho-family GTPases Rac and Cdc42. Our results indicate that signal transduction through Raf-1 depends on both Ras and the activation of the Pak pathway. As guanine-nucleotide-exchange activity on Rac can be stimulated by a Ras-dependent phosphatidylinositol-3-OH kinase, a mechanism could exist through which one Ras effector pathway can be influenced by another.