Translocation Animations

Protein translocation and biogenesis in the ER is a dynamic process that involves the coordinated function of complex machinery. For polytopic proteins this process involves multiple rounds of translocation initiation and termination, proper orientation of TM segments within respect to the membrane, and integration of TM helices into the lipid bilayer. All of these events must occur without mixing of ER lumenal and cytosolic components. We have generated several crude animations to illustrate how these events might be carried out by ER translocation machinery. Animations are controlled using buttons in lower right corner.

  1. Translocation of a secretory protein
  2. Translocation of a simple, Type I, transmembrane protein (Signal-Stop Transfer)
  3. Biogenesis of bitopic proteins by signal anchor sequences
  4. Polytopic protein biogenesis (simple cotranslational model)
  5. Aquaporin 1 biogenesis (topololgical maturation)
  6. Co- and post-translational translocation of CFTR N-terminus

SECRETORY PROTEINS are usually targeted to the ER when a signal sequence emerges from the ribosome and binds to the cytosolic signal recognition particle (SRP). SRP binding transiently pauses translation and brings the ribosome-nascent chain complex to the ER membrane via the SRP receptor. This facilitates ribosome binding to a protein conducting channel (translocon) composed of Sec61 alpha, beta, gamma, the TRanslocation Associated Membrane protein (TRAM) and other Translocation Associated Factors that include TRAP, Oligosaccharyltransferase, signal peptidase complex and others. As SRP releases the signal sequence it engages Sec61, and the translocon pore is opened by release of the lumenal chaperone BiP. In this manner, the elongating nascent chain exits the ribosome directly into the axial translocon pore. Concurrently, the ribosome-translocon junction establishes a seal that limits access of the nascent polypeptide to the cytosolic compartment. When translation is terminated, the polypeptide is released from the ribosome; the lumenal end of the translocon is resealed by BiP; and the translocon is available to accept another SRP-ribosome-nascent chain complex.

Three features of ER translocation are particularly noteworthy. First, translocation usually takes place while the nascent polypeptide is emerging from the large ribosomal subunit, i.e. translocation is both cotranslational and vectoral, proceeding from N- to C-terminus. Second, the nascent chain remains in an aqueous environment, shielded from membrane lipids during translocation. Third, the architecture of the ribosome translocon complex (RTC) is highly dynamic.

To play animation, click on the green arrow.

<img src="anim/protein_translocation.gif" width=350 height=280 border=0 />

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TRANSMEMBRANE PROTEIN

        Many bitopic (i.e. single-spanning) membrane proteins use an N-terminus signal sequence to initiate ER targeting and translocation events. However, integration of a TM segment into the ER membrane requires at least four additional steps. First, polypeptide translocation must be terminated. Second, the lumenal gate of the translocon must be closed. Third, the ribosome membrane junction must be disrupted to allow the growing nascent chain access to the cytosol. Fourth, the hydrophobic TM segment must exit laterally out of the translocon and into the lipid bilayer. These steps are directed by sequence determinants encoded within the nascent polypeptide called "stop transfer" (ST) sequences. ST sequences typically encompass the hydrophobic TM segment and its adjacent flanking residues. Two current theories describe how individual TM segments are integrated into the membrane. One proposes that the translocon is open laterally into the bilayer, and the TM segment partitions passively into the lipid phase via hydrophobic interactions. A second model predicts that integration occurs as a sequential process in which the TM segment passes through an ordered series of distinct protein environments. In the latter model,depicted below, full access to the lipid bilayer occurs only after the nascent chain is released from the ribosome at the completion of translation.

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<img src="anim/tm_protein.gif" width=350 height=280 border=0>

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        Bitopic membrane proteins can also be generated by a single sequence determinant called a "signal anchor" (SA) sequence. In addition to performing the combined functions of signal and ST sequences (i.e. targeting , translocon gating, translocation initiation and termination, and integration), SA sequences have the capacity to translocate either their C- or N-terminus flanking residues into the ER lumen and thus give rise to one of two different transmembrane topologies, type I (Nexo/Ccyto) or type II (Ncyto/Cexo). A critical difference between signal and SA sequences is that SA sequences are not cleaved and forms the TM segment. Structural features that determine SA translocation specificity include polar and charged residues flanking the TM segment, the length of the TM segment, and the rate of folding of N-terminus residues preceding the TM segment. While the precise mechanism remains unknown, SA translocation specificity is also likely controlled via interactions between the SA, the ribosome, Sec61, TRAM and/or other ER components.

To view, click on green arrow.

<img src="anim/sa_type_1.gif" width=280 height=250 border=0> <img src="anim/sa_type_II.gif" width=280 height=250 border=0>

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POLYTOPIC PROTEINS must be properly oriented within the translocon, integrated into the lipid bilayer, and finally assembled in the membrane. All of these events must occur efficiently and without mixing ER and cytosolic contents. Cells have adapted ER translocation machinery for this purpose, and many features of polytopic protein biogenesis thus parallel those of secretory and bitopic proteins.

The simplest model predicts that polytopic proteins are generated by a mechanisms similar to that used for simple , single-spanning proteins. In this process, topogenic determinants with signal or signal anchor activity target the nascent chain to the ER, open the translocon pore, and initiate translocation of the nascent polypeptide into the ER lumen. Synthesis of the next transmembrane (TM) segment sequence terminates translocation, closes the translocon, and directs the next loop of polypeptide into the cytosol. TM segments must also be released laterally from the translocon as they integrate into the lipid bilayer. Theoretically, this process could be repeated to generate proteins with any number of TM segments. Each topogenic determinant would thus direct the translocation and integration of TM segments in a vectoral and cotranslational manner from N-terminus to C-terminus as the nascent polypeptide emerged from membrane-bound ribosomes. This basic process is illustrated in the animation below. Note that the translocon channel must open into the ER lumen to allow passage of translocated polypeptide segments, but must also allow appropriate domains access to the cytosol. Thus the direction of protein movement is controlled by the configuration of the ribosome-translocon complex (RTC). The RTC is in turn controlled by the information encoded within topogenic determinants of the nascent polypeptide.

The animation below is based on gating properties described by the Johnson laboratory derived from fluorescence quenching of fluorophores incorporated into nascent polypeptides. Signal sequences and stop transfer sequences trigger alternate binding of the ribosome and the chaperone protein BIP to the cytosolic and lumenal surfaces of the translocon, respectively. However, the structural mechanisms that underlie these gating properties remain poorly understood. Major questions in polytopic protein biogenesis include: i) the timing with which multiple TM helices exit the translocon, ii) the mechanism by which TMs are transferred from the proteinaceous environment to the lipid environment, and iii) the mechanism and location by which helical packing and monomer folding take place. In the model below, each helix integrates sequentially and independently. While possible, this does not appear to be the case for all proteins.

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<img src="anim/tm_protein.gif" width=350 height=280 border=0>

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AQUAPORIN 1 BIOGENESIS involves a novel variation in the cotranslational folding model in which the initial topology of TM segments as they emerge from the ribosome differs from their final topology in the mature protein. This process is characterized by two key features. First, contrary to its expected behavior, TM2 lacks stop transfer activity and fails to terminate translocation. Second, AQP1 TM3 lacks signal sequence activity needed to translocate its C-terminus flanking residues. As a result of these properties, TM2 transiently passes into the ER lumen and TM3 initially acquires a Type I (Nexo/Ccyto) rather its mature than Type II (Ncyto/Cexo) topology. This behavior generates a folding intermediate that spans the membrane only four times. During and/or following synthesis of TM segments 4-6), however, additional folding information is provided that results in proper positioning of TM's 2-4. TM3 undergoes a 180° rotation about the plane of the membrane which correctly positions its adjacent TM segments, TM2 and TM4. Recent studies have demonstrated this change in topology is stabilized at least in part, by a hydrogen bond between Asn49 in TM2 and Asp185 in TM5. Ongoing studies are attempting to examine whether this reorientation occurs within the lipid bilayer, within the translocon proper, or at some intermediate location.

Topological reorientation events similar to that observed for AQP1 have been reported for engineered chimeras and may be a relatively common feature of native polytopic protein biogenesis.

In contrast to AQP1, AQP4 utilizes a simple cotranslational folding pathway described above. Topology of each TM segment is established independently and no reorientation of topology is needed. Specific residues at the N-terminus of TM2 and C-terminus of TM3 are responsible for differences between AQP1 and AQP4 folding pathways. Moreover, these folding pathways have functional implications because chimeric exchange of AQP4 and AQP1 residues that convert AQP1 to a cotranslational pathway completely disrupt AQP1 function. These results indicate that the repertoire of folding pathways permitted by ER translocation machinery accommodates greater sequence diversity than would be permitted if folding were limited to the strict cotranslational model shown above.

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<IMG SRC="anim/aqp1_biogen.gif" WIDTH=350 HEIGHT=280 BORDER=0>

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CFTR N-TERMINUS TOPOLOGY is established via co- and post-translational translcation events. TM1 and TM2 exhibit signal sequence activity capable of ER targeting and translocation with very inefficient due to the presence of two charged residues within its hydrophobic core. As a result, TM1 is able to direct correct topology in less than half of nascent CFT chains. By contrast, TM2 signal sequence activity is efficient and specific. Even in the absence of a functional TM1 signal sequence, TM2 is able to direct CFTR N-terminus topology via a ribosome dependent, posttranslational pathway. Note that in this latter pathway, TM1 together with the extracytoplasmic loop enter the translocon from a cytosolic orientation and that the direction of translolcatiaon proceeds in a C -> N terminus direction. This is consistent with TM2 acting as a Type II Signal Anchor sequence, and TM1 acting as a "posttranslational"

STOP TRANSFER
The topology derived from both pathways is the same but the sequence of events that gives rise to this topology depends on the particular functions of topogenic determinants. Thus a single functional signal sequence in either the first or second TM segment is sufficient for directing proper CFTR topology. This novel arrangement of topogenic information supports a model in which TM2 functions to ensure acorrect topology of CFTR in nascent chains that fail to translocate properly by TM1.

To play animation, click on the green arrow.

<img src="anim/cftr_cotranslational.gif" width=280 height=250 border=0 alt="CFTR co-translational"> <img src="anim/cftr_posttranslational.gif" width=280 height=250 border=0 alt="CFTR post-translational">

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