Tara Hessa

Recognition of transmembrane helices by the endoplasmic reticulum translocon

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.

Molecular code for transmembrane-helix recognition by the Sec61 translocon

Transmembrane α-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.

High-content genome-wide RNAi screens identify regulators of parkin upstream of mitophagy

An increasing body of evidence points to mitochondrial dysfunction as a contributor to the molecular pathogenesis of neurodegenerative diseases such as Parkinson’s disease. Recent studies of the Parkinson’s disease associated genes PINK1 (ref. 2) and parkin (PARK2, ref. 3) indicate that they may act in a quality control pathway preventing the accumulation of dysfunctional mitochondria. Here we elucidate regulators that have an impact on parkin translocation to damaged mitochondria with genome-wide small interfering RNA (siRNA) screens coupled to high-content microscopy. Screening yielded gene candidates involved in diverse cellular processes that were subsequently validated in low-throughput assays. This led to characterization of TOMM7 as essential for stabilizing PINK1 on the outer mitochondrial membrane following mitochondrial damage. We also discovered that HSPA1L (HSP70 family member) and BAG4 have mutually opposing roles in the regulation of parkin translocation. The screens revealed that SIAH3, found to localize to mitochondria, inhibits PINK1 accumulation after mitochondrial insult, reducing parkin translocation. Overall, our screens provide a rich resource to understand mitochondrial quality control.

Contribution of hydrophobic and electrostatic interactions to the membrane integration of the Shaker K+ channel voltage sensor domain

Membrane-embedded voltage-sensor domains in voltage-dependent potassium channels (Kv channels) contain an impressive number of charged residues. How can such highly charged protein domains be efficiently inserted into biological membranes? In the plant Kv 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 Kv 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 Kv channels and that the degree of cooperativity depends on a balance between electrostatic and hydrophobic forces.

The code for directing proteins for translocation across ER membrane: SRP cotranslationally recognizes specific features of a signal sequence

The signal recognition particle (SRP) cotranslationally recognizes signal sequences of secretory proteins and targets ribosome-nascent chain complexes 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 N(ε)-(5-azido-2 nitrobenzoyl)-Lys-tRNA(amb) 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.

Competition between neighboring topogenic signals during membrane protein insertion into the ER

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.

Competition between neighboring topogenic signals during membrane protein insertion into the ER: Kinetics of membrane protein insertion

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.

Stop-transfer efficiency of marginally hydrophobic segments depends on the length of the carboxy- terminal tail

Hydrophobic stop‐transfer sequences generally serve to halt the translocation of polypeptide chains across the endoplasmic reticulum membrane and become integrated as transmembrane α‐helices. Using engineered glycosylation sites as topology reporters, we show that the length of the nascent chain between a hydrophobic segment and the carboxy terminus of the protein can affect stop‐transfer efficiency. We also show that glycosylation sites located close to a proteins C terminus are modified in two distinct kinetic phases, one fast and one slow. Our findings suggest that membrane integration of a hydrophobic segment is not simply a question of thermodynamic equilibrium, but can be influenced by details of the translocation mechanism.

Measuring Endoplasmic Reticulum Signal Sequences Translocation Efficiency Using the Xbp1 Arrest Peptide

Secretory proteins translocate across the mammalian ER membrane co-translationally via the ribosome-sec61 translocation machinery. Signal sequences within the polypeptide, which guide this event, are diverse in their hydrophobicity, charge, length, and amino acid composition. Despite the known sequence diversity in the ER signals, it is generally assumed that they have a dominant role in determining co-translational targeting and translocation process. We have analyzed co-translational events experienced by secretory proteins carrying efficient versus inefficient signal sequencing, using an assay based on Xbp1 peptide-mediated translational arrest. With this method we were able to measure the functional efficiency of ER signal sequences. We show that an efficient signal sequence experiences a two-phase event whereby the nascent chain is pulled from the ribosome during its translocation, thus resuming translation and yielding full-length products. Conversely, the inefficient signal sequence experiences a single weaker pulling event, suggesting inadequate engagement by the translocation machinery of these marginally hydrophobic signal sequences.