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Docosahexaenoic acid (DHA) is a ω‐3 fatty acid typically obtained from the diet or endogenously synthesized through the action of elongases (ELOVLs) and desaturases. DHA is a key central nervous system constituent and the precursor of several molecules that regulate the resolution of inflammation. In the present study, we questioned whether the impaired synthesis of DHA affected neural plasticity and inflammatory status in the adult brain. To address this question, we investigated neural and inflammatory markers from mice deficient for ELOVL2 (Elovl2−/−), the key enzyme in DHA synthesis. From our findings, Elovl2−/− mice showed an altered expression of markers involved in synaptic plasticity, learning, and memory formation such as Egr‐1, Arc1, and BDNF specifically in the cerebral cortex, impacting behavioral functions only marginally. In parallel, we also found that DHA‐deficient mice were characterized by an increased expression of pro‐inflammatory molecules, namely TNF, IL‐1β, iNOS, caspase‐1 as well as the activation and morphologic changes of microglia in the absence of any brain injury or disease. Reintroducing DHA in the diet of Elovl2−/− mice reversed such alterations in brain plasticity and inflammation. Hence, impairment of systemic DHA synthesis can modify the brain inflammatory and neural plasticity status, supporting the view that DHA is an essential fatty acid with an important role in keeping inflammation within its physiologic boundary and in shaping neuronal functions in the central nervous system.

Transmembrane alpha-helices in integral membrane proteins are recognized co-translationally and inserted into the membrane of the endoplasmic reticulum by the Sec61 translocon. A full quantitative description of this phenomenon, linking amino acid sequence to membrane insertion efficiency, is still lacking. Here, using in vitro translation of a model protein in the presence of dog pancreas rough microsomes to analyse a large number of systematically designed hydrophobic segments, we present a quantitative analysis of the position-dependent contribution of all 20 amino acids to membrane insertion efficiency, as well as of the effects of transmembrane segment length and flanking amino acids. The emerging picture of translocon-mediated transmembrane helix assembly is simple, with the critical sequence characteristics mirroring the physical properties of the lipid bilayer.

Membrane-embedded voltage-sensor domains in voltage-dependent potassium channels (K v channels) contain an impressive number of charged residues. How can such highly charged protein domains be efficiently inserted into biological membranes? In the plant K v channel KAT1, the S2, S3, and S4 transmembrane helices insert cooperatively, because the S3, S4, and S3–S4 segments do not have any membrane insertion ability by themselves. Here we show that, in the Drosophila Shaker K v channel, which has a more hydrophobic S3 helix than KAT1, S3 can both insert into the membrane by itself and mediate the insertion of the S3–S4 segment in the absence of S2. An engineered KAT1 S3–S4 segment in which the hydrophobicity of S3 was increased or where S3 was replaced by Shaker S3 behaves as Shaker S3–S4. Electrostatic interactions among charged residues in S2, S3, and S4, including the salt bridges between E283 or E293 in S2 and R368 in S4, are required for fully efficient membrane insertion of the Shaker voltage-sensor domain. These results suggest that cooperative insertion of the voltage-sensor transmembrane helices is a property common to K v channels and that the degree of cooperativity depends on a balance between electrostatic and hydrophobic forces.

Membrane proteins depend on complex translocation machineries for insertion into target membranes. Although it has long been known that an abundance of nonpolar residues in transmembrane helices is the principal criterion for membrane insertion, the specific sequence-coding for transmembrane helices has not been identified. By challenging the endoplasmic reticulum Sec61 translocon with an extensive set of designed polypeptide segments, we have determined the basic features of this code, including a 'biological' hydrophobicity scale. We find that membrane insertion depends strongly on the position of polar residues within transmembrane segments, adding a new dimension to the problem of predicting transmembrane helices from amino acid sequences. Our results indicate that direct protein-lipid interactions are critical during translocon-mediated membrane insertion.

The signal recognition particle (SRP) cotranslationally recognizes signal sequences of secretory proteins and targets ribosome-nascent chain complexes (RNCs) to the SRP receptor in the endoplasmic reticulum membrane, initiating translocation of the nascent chain through the Sec61 translocon. Although signal sequences do not have homology they have similar structural regions: a positively charged N-terminus, a hydrophobic core and a more polar C-terminal region that contains the cleavage site for the signal peptidase. Here, we have used site-specific photocrosslinking to study SRP-signal sequence interactions. A photoreactive probe was incorporated into the middle of wild-type or mutated signal sequences of the secretory protein preprolactin by in vitro translation of mRNAs containing an amber stop codon in the signal peptide in the presence of the Nε-(5-azido-2 nitrobenzoyl)-Lys-tRNAamb amber suppressor. A homogeneous population of SRP-ribosome-nascent chain complexes was obtained by the use of truncated mRNAs in translations performed in the presence of purified canine SRP. Quantitative analysis of the photoadducts revealed that charged residues at the N-terminus of the signal sequence or in the early part of the mature protein have only a mild effect on the SRP-signal sequence association. However, deletions of amino acid residues in the hydrophobic portion of the signal sequence severely affect SRP binding. The photocrosslinking data correlate with targeting efficiency and translocation across the membrane. Thus, the hydrophobic core of the signal sequence is primarily responsible for its recognition and binding by SRP, while positive charges fine-tune the SRP-signal sequence affinity and targeting to the translocon.

A substantial proportion of the genome encodes membrane proteins that are delivered to the endoplasmic reticulum by dedicated targeting pathways. Membrane proteins that fail targeting must be rapidly degraded to avoid aggregation and disruption of cytosolic protein homeostasis. The mechanisms of mislocalized protein (MLP) degradation are unknown. Here we reconstitute MLP degradation in vitro to identify factors involved in this pathway. We find that nascent membrane proteins tethered to ribosomes are not substrates for ubiquitination unless they are released into the cytosol. Their inappropriate release results in capture by the Bag6 complex, a recently identified ribosome-associating chaperone. Bag6-complex-mediated capture depends on the presence of unprocessed or non-inserted hydrophobic domains that distinguish MLPs from potential cytosolic proteins. A subset of these Bag6 complex 'clients' are transferred to TRC40 for insertion into the membrane, whereas the remainder are rapidly ubiquitinated. Depletion of the Bag6 complex selectively impairs the efficient ubiquitination of MLPs. Thus, by its presence on ribosomes that are synthesizing nascent membrane proteins, the Bag6 complex links targeting and ubiquitination pathways. We propose that such coupling allows the fast tracking of MLPs for degradation without futile engagement of the cytosolic folding machinery.

To better define the mechanism of membrane protein insertion into the membrane of the endoplasmic reticulum, we measured the kinetics of translocation across microsomal membranes of the N-terminal lumenal tail and the lumenal domain following the second transmembrane segment (TM2) in the multispanning mouse protein Cig30. In the wild-type protein, the N-terminal tail translocates across the membrane before the downstream lumenal domain. Addition of positively charged residues to the N-terminal tail dramatically slows down its translocation and allows the downstream lumenal domain to translocate at the same time as or even before the N-tail. When TM2 is deleted, or when the loop between TM1 and TM2 is lengthened, addition of positively charged residues to the N-terminal tail causes TM1 to adopt an orientation with its N-terminal end in the cytoplasm. We suggest that the topology of the TM1-TM2 region of Cig30 depends on a competition between TM1 and TM2 such that the transmembrane segment that inserts first into the ER membrane determines the final topology.