Post-translational targeting of a tail-anchored green fluorescent protein to the endolpasmic reticulum

Microsomal aldehyde dehydrogenase (msALDH) is a tail-anchored protein localized to the cytoplasmic face of the endoplasmic reticulum (ER). The carboxyl-terminal 35 amino acids of msALDH possess ER-targeting sequences in addition to a hydrophobic membrane-spanning domain. To study the mechanism for ER targeting of this protein in vivo, we took advantage of a green fluorescent protein-msALDH fusion protein containing the last 35 amino acids of msALDH [GFPALDH(35)]. When expressed from cDNA in COS-7 cells, the fusion protein was localized to the ER. We then prepared a recombinant fusion protein and injected it into the cytoplasm of COS-7 cells. The injected protein was correctly localized to the ER after a 30-min incubation at 37 degrees C. However, a recombinant fusion protein that contained only the transmembrane domain of msALDH failed to be targeted to the ER. When the assay was carried out at 4 degrees C, the recombinant GFPALDH(35) remained in the cytoplasm. Moreover, incubation of COS-7 cells under conditions of ATP depletion resulted in the cytoplasmic distribution of the injected protein. These results indicate that GFPALDH(35) is targeted to the ER post-translationally via an ATP-dependent pathway. This microinjection system worked effectively in different mammalian cell types, suggesting a common mechanism for ER targeting of the tail-anchored protein.     

Two hydrophobic segments of the RTN1 family determine the ER localization and retention

Reticulon (RTN) proteins are localized to the endoplasmic reticulum (ER), and are related to intracellular membrane trafficking, apoptosis, inhibiting axonal regeneration, and Alzheimer's disease. The RTN proteins are produced without an N-terminal signal peptide. Their C-terminal domain contains two long hydrophobic segments. We analyzed the ER localization signal of human RTN1-A. Mutant proteins lacking the first (39 residues) or second (36 residues) hydrophobic segment showed ER localization. On the other hand, the mutant lacking both hydrophobic segments was cytosolic. Enhanced green fluorescent protein (EGFP) tagged with the first or second hydrophobic segment of RTN1-A was localized to the ER. These results suggest that each hydrophobic segment determines the ER localization. In addition, EGFP tagged with the truncated form of the first hydrophobic segment exhibited the localization to the Golgi rather than the ER. This suggests that the length of the hydrophobic segment contributes to the ER retention of RTN1-A.     

The stop transfer sequence of the human UDP-glucuronosyltransferase 1A determines localization to the endoplasmic reticulum by both static retention and retrieval mechanisms

Human UDP-glucuronosyltransferase 1A (UGT1A) isoforms are endoplasmic reticulum (ER)-resident type I membrane proteins responsible for the detoxification of a broad range of toxic phenolic compounds. These proteins contain a C-terminal stop transfer sequence with a transmembrane domain (TMD), which anchors the protein into the membrane, followed by a short cytosolic tail (CT). Here, we investigated the mechanism of ER residency of UGT1A mediated by the stop transfer sequence by analysing the subcellular localization and sensitivity to endoglycosidases of chimeric proteins formed by fusion of UGT1A stop transfer sequence (TMD/CT) with the ectodomain of the plasma membrane CD4 reporter protein. We showed that the stop transfer sequence, when attached to C-terminus of the CD4 ectodomain was able to prevent it from being transported to the cell surface. The protein was retained in the ER indicating that this sequence functions as an ER localization signal. Furthermore, we demonstrated that ER localization conferred by the stop transfer sequence was mediated in part by the KSKTH retrieval signal located on the CT. Interestingly, our data indicated that UGT1A TMD alone was sufficient to retain the protein in ER without recycling from Golgi compartment, and brought evidence that organelle localization conferred by UGT1A TMD was determined by the length of its hydrophobic core. We conclude that both retrieval mechanism and static retention mediated by the stop transfer sequence contribute to ER residency of UGT1A proteins.     

Switching the sorting mode of membrane proteins from cotranslational endoplasmic reticulum targeting to posttranslational mitochondrial import

Hydrophobic membrane proteins are cotranslationally targeted to the endoplasmic reticulum (ER) membrane, mediated by hydrophobic signal sequence. Mitochondrial membrane proteins escape this mechanism despite their hydrophobic character. We examined sorting of membrane proteins into the mitochondria, by using mitochondrial ATP-binding cassette (ABC) transporter isoform (ABC-me). In the absence of 135-residue N-terminal hydrophilic segment (N135), the membrane domain was integrated into the ER membrane in COS7 cells. Other sequences that were sufficient to import soluble protein into mitochondria could not import the membrane domain. N135 imports other membrane proteins into mitochondria. N135 prevents cotranslational targeting of the membrane domain to ER and in turn achieves posttranslational import into mitochondria. In a cell-free system, N135 suppresses targeting to the ER membranes, although it does not affect recognition of hydrophobic segments by signal recognition particle. We conclude that the N135 segment blocks the ER targeting of membrane proteins even in the absence of mitochondria and switches the sorting mode from cotranslational ER integration to posttranslational mitochondrial import.     

Determinants of the enzymatic activity and the subcellular localization of aspartate N-acetyltransferase

Aspartate N-acetyltransferase (NAT8L, N-acetyltransferase 8-like), the enzyme that synthesizes N-acetylaspartate, is membrane-bound and is at least partially associated with the ER (endoplasmic reticulum). The aim of the present study was to determine which regions of the protein are important for its catalytic activity and its subcellular localization. Transfection of truncated forms of NAT8L into HEK (human embryonic kidney)-293T cells indicated that the 68 N-terminal residues (regions 1 and 2) have no importance for the catalytic activity and the subcellular localization of this enzyme, which was exclusively associated with the ER. Mutation of conserved residues that precede (Arg81 and Glu101, in region 3) or follow (Asp168 and Arg220, in region 5) the putative membrane region (region 4) markedly affected the kinetic properties, suggesting that regions 3 and 5 form the catalytic domain and that the membrane region has a loop structure. Evidence is provided for the membrane region comprising α-helices and the catalytic site being cytosolic. Transfection of chimaeric proteins in which GFP (green fluorescent protein) was fused to different regions of NAT8L indicated that the membrane region (region 4) is necessary and sufficient to target NAT8L to the ER. Thus NAT8L is targeted to the ER membrane by a hydrophobic loop that connects two regions of the catalytic domain.     

Retention of subunits of the oligosaccharyltransferase complex in the endoplasmic reticulum

Membrane proteins of the endoplasmic reticulum (ER) may be localized to this organelle by mechanisms that involve retention, retrieval, or a combination of both. For luminal ER proteins, which contain a KDEL domain, and for type I transmembrane proteins carrying a dilysine motif, specific retrieval mechanisms have been identified. However, most ER membrane proteins do not contain easily identifiable retrieval motifs. ER localization information has been found in cytoplasmic, transmembrane, or luminal domains. In this study, we have identified ER localization domains within the three type I transmembrane proteins, ribophorin I (RI), ribophorin II (RII), and OST48. Together with DAD1, these membrane proteins form an oligomeric complex that has oligosaccharyltransferase (OST) activity. We have previously shown that ER retention information is independently contained within the transmembrane and the cytoplasmic domain of RII, and in the case of RI, a truncated form consisting of the luminal domain was retained in the ER. To determine whether other domains of RI carry additional retention information, we have generated chimeras by exchanging individual domains of the Tac antigen with the corresponding ones of RI. We demonstrate here that only the luminal domain of RI contains ER retention information. We also show that the dilysine motif in OST48 functions as an ER localization motif because OST48 in which the two lysine residues are replaced by serine (OST48ss) is no longer retained in the ER and is found instead also at the plasma membrane. OST48ss is, however, retained in the ER when coexpressed with RI, RII, or chimeras, which by themselves do not exit from the ER, indicating that they may form partial oligomeric complexes by interacting with the luminal domain of OST48. In the case of the Tac chimera containing only the luminal domain of RII, which by itself exits from the ER and is rapidly degraded, it is retained in the ER and becomes stabilized when coexpressed with OST48.     

The juxtamembrane sequence of cytochrome P-450 2C1 contains an endoplasmic reticulum retention signal

The N-terminal signal anchor of cytochrome P-450 2C1 mediates retention in the endoplasmic reticulum (ER) membrane of several reporter proteins. The same sequence fused to the C terminus of the extracellular domain of the epidermal growth factor receptor permits transport of the chimeric protein to the plasma membrane. In the N-terminal position, the ER retention function of this signal depends on the polarity of the hydrophobic domain and the sequence KQS in the short hydrophilic linker immediately following the transmembrane domain. To determine what properties are required for the ER retention function of the signal anchor in a position other than the N terminus, the effect of mutations in the linker and hydrophobic domains on subcellular localization in COS1 cells of chimeric proteins with the P-450 signal anchor in an internal or C-terminal position was analyzed. For the C-terminal position, the signal anchor was fused to the end of the luminal domain of epidermal growth factor receptor, and green fluorescent protein was additionally fused at the C terminus of the signal anchor for the internal position. In these chimeras, the ER retention function of the signal anchor was rescued by deletion of three leucines at the C-terminal side of its hydrophobic domain; however, deletion of three valines from the N-terminal side did not affect transport to the cell surface. ER retention of the C-terminal deletion mutants was eliminated by substitution of alanines for glutamine and serine in the linker sequence. These data are consistent with a model in which the position of the linker sequence at the membrane surface, which is critical for ER retention, is dependent on the transmembrane domain.     

Molecular cloning, sequencing, and expression of cDNA for rat liver microsomal aldehyde dehydrogenase.

The cDNA clone for rat liver microsomal aldehyde dehydrogenase (msALDH) was isolated and sequenced. The deduced amino acid sequence consisting of 484 amino acid residues revealed that the carboxyl-terminal region of msALDH has a hydrophobic segment, which is probably important for the insertion of this enzyme into the endoplasmic reticulum membrane. COS-1 cells transfected with the expression vector pcD containing the full-length cDNA showed that the active enzyme was expressed and localized mainly on the cytoplasmic surface of the endoplasmic reticulum membranes. It has been proposed that ALDH isozymes form a superfamily consisting of class 1, 2, and 3 ALDHs (Hempel, J., Harper, K., and Lindahl, R., (1989) Biochemistry 28, 1160-1167). Comparison of the amino acid sequence of rat liver msALDH with those of rat other class ALDHs showed that msALDH was 24.2, 24.0, and 65.5% identical to phenobarbital-inducible ALDH (variant class 1), mitochondrial ALDH (class 2), and tumor-associated ALDH (class 3), respectively. Several amino acid residues common to the other known ALDHs, however, were found to be conserved in msALDH. Based on these results, we proposed to classify msALDH as a new type, class 4 ALDH.     

Membrane localization of cyclic nucleotide phosphodiesterase 3 (PDE3). Two N-terminal domains are required for the efficient targeting to, and association of, PDE3 with endoplasmic reticulum

Subcellular localization of cyclic nucleotide phosphodiesterases (PDEs) may be important in compartmentalization of cAMP/cGMP signaling responses. In 3T3-L1 adipocytes, mouse (M) PDE3B was associated with the endoplasmic reticulum (ER) as indicated by its immunofluorescent colocalization with the ER protein BiP and subcellular fractionation studies. In transfected NIH 3006 or COS-7 cells, recombinant wild-type PDE3A and PDE3B isoforms were both found almost exclusively in the ER. The N-terminal portion of PDE3 can be arbitrarily divided into region 1 (aa 1-300), which contains a large hydrophobic domain with six predicted transmembrane helices, followed by region 2 (aa 301-500) containing a smaller hydrophobic domain (of approximately 50 aa). To investigate the role of regions 1 and 2 in membrane association, we examined the subcellular localization of a series of catalytically active, Flag-tagged N-terminal-truncated human (H) PDE3A and MPDE3B recombinants, as well as a series of fragments from regions 1 and 2 of MPDE3B synthesized as enhanced green fluorescent (EGFP) fusion proteins in COS-7 cells. In COS-7 cells, the localization of a mutant HPDE3A, lacking the first 189 amino acids (aa) and therefore four of the six predicted transmembrane helices (H3A-Delta189), was virtually identical to that of the wild type. M3B-Delta302 (lacking region 1) and H3A-Delta397 (lacking region 1 as well as part of region 2) retained, to different degrees, the ability to associate with membranes, albeit less efficiently than H3A-Delta189. Proteins that lacked both regions 1 and 2, H3A-Delta510 and M3B-Delta604, did not associate with membranes. Consistent with these findings, region 1 EGFP-MPDE3B fusion proteins colocalized with the ER, whereas region 2 EGFP fusion proteins were diffusely distributed. Thus, some portion of the N-terminal hydrophobic domain in region 1 plus a second domain in region 2 are important for efficient membrane association/targeting of PDE3.     

The luminal N-terminus of yeast Nvj1 is an inner nuclear membrane anchor

The endoplasmic reticulum (ER) in Saccharomyces cerevisiae is largely divided between perinuclear and cortical compartments. Yeast Nvj1 localizes exclusively to small patches on the perinuclear ER where it interacts with Vac8 in the vacuole membrane to form nucleus-vacuole (NV) junctions. Three regions of Nvj1 mediate the biogenesis of NV junctions. A membrane-spanning domain targets the protein to the ER. The C-terminus binds Vac8 in the vacuole membrane, which induces the clustering of both proteins into NV junctions. The luminal N-terminus is required for strict perinuclear localization. Three-dimensional cryo-electron tomography reveals that Nvj1 clamps the separation between the two nuclear membranes to half the width of bulk nuclear envelope. The N-terminus contains a hydrophobic sequence bracketed by basic residues that resembles outer mitochondrial membrane signal-anchors. The hydrophobic sequence can be scrambled or reversed without affecting function. Mutations that reduce the hydrophobicity of the core sequence or affect the distribution of basic residues cause mislocalization to the cortical ER. We conclude that the N-terminus of Nvj1 is a retention sequence that bridges the perinuclear lumen and inserts into the inner nuclear membrane.     

Targeting of tail‐anchored proteins to Trichomonas vaginalis hydrogenosomes

Tail‐anchored (TA) proteins are membrane proteins that are found in all domains of life. They consist of an N‐terminal domain that performs various functions and a single transmembrane domain (TMD) near the C‐terminus. In eukaryotes, TA proteins are targeted to the membranes of mitochondria, the endoplasmic reticulum (ER), peroxisomes and in plants, chloroplasts. The targeting of these proteins to their specific destinations correlates with the properties of the C‐terminal domain, mainly the TMD hydrophobicity and the net charge of the flanking regions. Trichomonas vaginalis is a human parasite that has adapted to oxygen‐poor environment. This adaptation is reflected by the presence of highly modified mitochondria (hydrogenosomes) and the absence of peroxisomes. The proteome of hydrogenosomes is considerably reduced; however, our bioinformatic analysis predicted 120 putative hydrogenosomal TA proteins. Seven proteins were selected to prove their localization. The elimination of the net positive charge in the C‐tail of the hydrogenosomal TA4 protein resulted in its dual localization to hydrogenosomes and the ER, causing changes in ER morphology. Domain mutation and swap experiments with hydrogenosomal (TA4) and ER (TAPDI) proteins indicated that the general principles for specific targeting are conserved across eukaryotic lineages, including T. vaginalis; however, there are also significant lineage‐specific differences.     

Recognition of the carboxyl-terminal signal for GPI modification requires translocation of its hydrophobic domain across the ER membrane

A carboxyl-terminal hydrophobic domain is an essential component of the processed signal for attachment of the glycosyl-phosphatidylinositol (GPI) membrane anchor to proteins and it is linked to the site (omega) of GPI modification by a spacer domain. This study was designed to test the hypothesis that the hydrophobic domain interacts with the lipid bilayer of the endoplasmic reticulum (ER) membrane to optimally position the omega site for GPI modification. The hydrophobic domain of the GPI signal in the human folate receptor (FR) type alpha was substituted with the carboxyl-terminal segment of the low-density lipoprotein receptor (LDLR), including its membrane spanning region, without altering either the spacer or the omega site. The FR-alpha/LDLR chimera was not GPI modified but was attached to the plasma membrane by a polypeptide anchor. When the carboxyl-terminal half of the hydrophobic transmembrane polypeptide in the FR-alpha/LDLR chimera was altered by introduction of negatively charged (Asp) residues, or when the cytosolic domain in the chimera was deleted, the mutated proteins became GPI-anchored. On the other hand, attachment of a carboxyl-terminal segment of LDLR including the entire cytosolic domain to FR-alpha converted it into a transmembrane protein. The results indicate that in the FR-alpha/LDLR chimera the inability of the cellular machinery for GPI modification to recognize the hydrophobic domain is not due to the intrinsic nature of the peptide, but is rather due to the retention of the peptide within the lipid bilayer. It follows that the hydrophobic domain in the signal for GPI modification must traverse the ER membrane prior to recognition of the omega site by the GPI-protein transamidase. The results thus establish a critical topographical requirement for recognition of the GPI signal in the ER.     

70-kDa peroxisomal membrane protein related protein (P70R/ABCD4) localizes to endoplasmic reticulum not peroxisomes, and NH2-terminal hydrophobic property determines the subcellular localization of ABC subfamily D proteins

70-kDa peroxisomal membrane protein related protein (P70R/ABCD4) is a member of ATP-binding cassette (ABC) protein subfamily D. ABC subfamily D proteins are also known as peroxisomal ABC proteins. Therefore, P70R is thought to be a peroxisomal membrane protein. However, the subcellular localization of P70R is not extensively investigated. In this study, we transiently expressed P70R in fusion with HA (P70R-HA) in CHO cells and examined subcellular localization by immunofluorescence. Surprisingly, P70R-HA was localized to the endoplasmic reticulum (ER), not to peroxisomes. To examine the ER-targeting property of P70R, we expressed various NH₂-terminal deletion constructs of P70R. Among the NH₂-terminal deletion constructs, mutant proteins starting with hydrophobic transmembrane segment (TMS) were localized to ER, but the ones containing the NH₂-terminal hydrophilic cytosolic domain were not. ABC subfamily D proteins destined for peroxisomes have NH₂-terminal hydrophilic region adjacent to TMS1. However, only P70R lacks the region and is translated with NH₂-terminal hydrophobic TMS1. Furthermore, attachment of the NH₂-terminal hydrophilic domain to the NH₂-terminus of P70R excluded P70R from the ER-targeting pathway. These data suggest that P70R resides in the ER but not the peroxisomal membranes, and the hydrophobic property of NH₂-terminal region determines the subcellular localization of ABC subfamily D proteins.     

Subcellular localization and targeting of N-acetylglucosaminyl phosphatidylinositol de-N-acetylase, the second enzyme in the glycosylphosphatidylinositol biosynthetic pathway

The second step in glycosylphosphatidylinositol biosynthesis is the de-N-acetylation of N-acetylglucosaminylphosphatidylinositol (GlcNAc-PI) catalyzed by N-acetylglucosaminylphosphatidylinositol deacetylase (PIG-L). Previous studies of mouse thymoma cells showed that GlcNAc-PI de-N-acetylase activity is localized to the endoplasmic reticulum (ER) but enriched in a mitochondria-associated ER membrane (MAM) domain. Because PIG-L has no readily identifiable ER sorting determinants, we were interested in learning how PIG-L is localized to the ER and possibly enriched in MAM. We used HeLa cells transiently or stably expressing epitope-tagged PIG-L variants or chimeric constructs composed of elements of PIG-L fused to Tac antigen, a cell surface protein. We first analyzed the subcellular distribution of PIG-L and Glc-NAc-PI-de-N-acetylase activity and then studied the localization of Tac-PIG-L chimeras to identify sequence elements in PIG-L responsible for its subcellular localization. We show that human PIG-L is a type I membrane protein with a large cytoplasmic domain and that, unlike the result with mouse thymoma cells, both PIG-L and GlcNAc-PI-de-N-acetylase activity are uniformly distributed between ER and MAM in HeLa cells. Analyses of a series of Tac-PIG-L chimeras indicated that PIG-L contains two ER localization signals, an independent retention signal located between residues 60 and 88 of its cytoplasmic domain and another weak signal in the luminal and transmembrane domains that functions autonomously in the presence of membrane proximal residues of the cytoplasmic domain that themselves lack any retention information. We conclude that PIG-L, like a number of other ER membrane proteins, is retained in the ER through a multi-component localization signal rather than a discrete sorting motif.     

The amino-terminal domain of the lamin B receptor is a nuclear envelope targeting signal

The lamin B receptor (LBR) is a polytopic protein of the inner nuclear membrane. It is synthesized without a cleavable amino-terminal signal sequence and composed of a nucleoplasmic amino-terminal domain of 204 amino acids followed by a hydrophobic domain with eight putative transmembrane segments. To identify a nuclear envelope targeting signal, we have examined the cellular localization by immunofluorescence microscopy of chicken LBR, its amino-terminal domain and chimeric proteins transiently expressed in transfected COS-7. Full- length LBR was targeted to the nuclear envelope. The amino-terminal domain, without any transmembrane segments, was transported to the nucleus but excluded from the nucleolus. When the amino-terminal domain of LBR was fused to the amino-terminal side of a transmembrane segment of a type II integral membrane protein of the ER/plasma membrane, the chimeric protein was targeted to the nuclear envelope, likely the inner nuclear membrane. When the amino-terminal domain was deleted from LBR and replaced by alpha-globin, the chimeric protein was retained in the ER. These findings demonstrate that the amino-terminal domain of LBR is targeted to the nucleus after synthesis in the cytoplasm and that this polypeptide can function as a nuclear envelope targeting signal when located at the amino terminus of a type II integral membrane protein synthesized on the ER.     

Molecular determinants of the subcellular localization of the Drosophila Bcl-2 homologues DEBCL and BUFFY

The Bcl-2-family of proteins localize to intracellular membranes via a C-terminal hydrophobic membrane anchor (MA) domain, to exert their antiapoptotic or proapoptotic functions. In Drosophila, both Bcl-2 family members, DEBCL and BUFFY, contain an MA. In DEBCL the MA is necessary for the localization of protein to mitochondria and for its proapoptotic activity. BUFFY is highly similar to DEBCL but its localization and function are not clearly defined. Here, we report on comparative analysis of BUFFY and DEBCL to decipher the molecular basis for their subcellular localization. We show that these two proteins localize to distinct intracellular membranes, DEBCL predominantly to mitochondria and BUFFY to endoplasmic reticula (ER). Our results suggest that the MA-flanking residues in DEBCL, homologous to Bcl-X(L), are required for the targeting of DEBCL to mitochondria. The C-terminal positively charged residues present in DEBCL are absent in BUFFY, which allows for its localization to ER. The MA in both proteins is required for the correct targeting and proapoptotic activities of these proteins. Interestingly, a functional nuclear localization signal was identified in the N-terminal region of BUFFY and in the absence of the MA, BUFFY accumulated in the nucleus. The functional implications of these findings are discussed.     

Deletions into an NH2-terminal hydrophobic domain result in secretion of rotavirus VP7, a resident endoplasmic reticulum membrane glycoprotein

Rotavirus, a non-enveloped reovirus, buds into the rough endoplasmic reticulum and transiently acquires a membrane. The structural glycoprotein, VP7, a 38-kD integral membrane protein of the endoplasmic reticulum (ER), presumably transfers to virus in this process. The gene for VP7 potentially encodes a protein of 326 amino acids which has two tandem hydrophobic domains at the NH2-terminal, each preceded by an in-frame ATG codon. A series of deletion mutants constructed from a full-length cDNA clone of the Simian 11 rotavirus VP7 gene were expressed in COS 7 cells. Products from wild-type, and mutants which did not affect the second hydrophobic domain of VP7, were localized by immunofluorescence to elements of the ER only. However, deletions affecting the second hydrophobic domain (mutants 42-61, 43-61, 47-61) showed immunofluorescent localization of VP7 which coincided with that of wheat germ agglutinin, indicating transport to the Golgi apparatus. Immunoprecipitable wild-type protein, or an altered protein lacking the first hydrophobic sequence, remained intracellular and endo-beta-N-acetylglucosaminidase H sensitive. In contrast, products of mutants 42-61, 43-61, and 47-61 were transported from the ER, and secreted. Glycosylation of the secreted molecules was inhibited by tunicamycin, resistant to endo-beta-N-acetylglucosaminidase H digestion and therefore of the N-linked complex type. An unglycosylated version of VP7 was also secreted. We suggest that the second hydrophobic domain contributes to a positive signal for ER location and a membrane anchor function. Secretion of the mutant glycoprotein implies that transport can be constitutive with the destination being dictated by an overriding compartmentalization signal.     

Monotopic topology is required for lipid droplet targeting of ancient ubiquitous protein 1

Ancient ubiquitous protein 1 (AUP1) is a multifunctional protein, which acts on both lipid droplets (LDs) and the endoplasmic reticulum (ER) membrane. Double localization to these two organelles, featuring very different membrane characteristics, was observed also for several other integral proteins, but little is known about the signals and mechanisms behind dual protein targeting to ER and LDs. Here we dissect the AUP1 targeting signals by analyses of localization and topology of several deletion and point mutants. We found that AUP1 is inserted into the membrane of the ER in a monotopic hairpin fashion, and subsequently transported to the hemi-membrane of LDs. A single domain localized in the N-terminal part of AUP1 enables its ER residence, the monotopic insertion, and the LD localization. Different specific residues within this multifunctional domain are responsible for achieving the complex spatial distribution pattern. A mutation of three amino acids, which changes AUP1 topology from hairpin to transmembrane, abolishes LD localization. These findings suggest that the cell is able to target a protein to multiple intracellular locations using a single domain.     

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.     

Location of sequences within rotavirus SA11 glycoprotein VP7 which direct it to the endoplasmic reticulum.

The Simian 11 rotavirus glycoprotein VP7 is directed to the endoplasmic reticulum (ER) of the cell and retained as an integral membrane protein. The gene coding for VP7 predicts two potential initiation codons, each of which precedes a hydrophobic region of amino acids (H1 and H2) with the characteristics of a signal peptide. Using the techniques of gene mutagenesis and expression, we have determined that either hydrophobic domain alone can direct VP7 to the ER. A protein lacking both hydrophobic regions was not transported to the ER. Some polypeptides were directed across the ER membrane and then into the secretory pathway of the cell. For a variant retaining only the H1 domain, secretion was cleavage dependent, since an amino acid change which prevented cleavage also stopped secretion. However, secretion of two other deletion mutants lacking H1 and expressing truncated H2 domains was unaffected by this mutation, suggesting that these proteins were secreted without cleavage of their NH2-terminal hydrophobic regions or secreted after cleavage at a site(s) not predicted by current knowledge.