Membrane Insertion Kinetics of a Protein Domain In Vivo

Abstract

The pathway by which segments of a polytopic membrane protein are inserted into the membrane has not been resolved in vivo. We have developed an in vivo kinetic assay to examine the insertion pathway of the polytopic protein bacterioopsin, the apoprotein of Halobacterium salinarum bacteriorhodopsin. Strains were constructed that express the bacteriorhodopsin mutants I4C:H 6 and T5C:H 6 , which carry a unique Cys in the N-terminal extracellular domain and a polyhistidine tag at the C terminus. Translocation of the N-terminal domain was detected using a membrane-impermeant gel shift reagent to derivatize the Cys residue of nascent radiolabeled molecules. Derivatization was assessed by gel electrophoresis of the fully elongated radiolabeled population. The time required to translocate and fully derivatize the Cys residues of I4C:H 6 and T5C:H 6 is 46 ؎ 9 and 61 ؎ 6 s, respectively. This is significantly shorter than the elongation times of the proteins, which are 114 ؎ 26 and 169 ؎ 16 s, respectively. These results establish that translocation of the bacterioopsin N terminus and insertion of the first transmembrane segment occur co-translationally and confirm the use of the assay to monitor the kinetics of polytopic membrane protein insertion in vivo.An essential step in the biogenesis of polytopic or multispanning membrane proteins is the insertion of polypeptide segments into the membrane. To elucidate the insertion mechanism in vivo, the cellular factors that participate in this process must be characterized, and the pathway or sequence with which the polypeptide segments are inserted must be determined.Cellular factors have been identified that mediate protein insertion into the eukaryotic endoplasmic reticulum (1, 2) and bacterial cytoplasmic membranes (3-5). These include the signal recognition particle (SRP), 1 a cytosolic ribonucleoprotein complex, and the secretory translocase, a membrane protein complex. SRP directs ribosome-bound nascent polypeptides to the secretory translocase, which in eukaryotes forms an aqueous channel into which the polypeptides are inserted (6, 7). Sequence conservation of SRP and secretory translocase subunits (8, 9) suggests that aspects of the insertion mechanism are universal.Less is known about the insertion pathway of polytopic membrane proteins. In eukaryotes, a co-translational, sequential insertion pathway (10, 11) is favored (12). In support of this model, the insertion of transmembrane segments of polytopic membrane proteins has been shown to be mechanistically coupled to translation (13-15) and to occur sequentially (15-17). However, other evidence contradicts this model. At least one eukaryotic polytopic membrane protein appears to insert posttranslationally (18), and several others exhibit multiple topologies that may reflect a nonsequential insertion pathway (19 -21). In bacteria, it is not known whether insertion is cotranslational and sequential or more closely resembles protein secretion, where translocation is independent from elongation (22, 23). There is evid...

Ordered membrane insertion of an archaeal opsin in vivo

“…In accord with this hypothesis, the RNA component of the signal recognition particle is localized to the membrane on induction of BO synthesis (19). The model also is supported by our previous work, in which we used an in vivo kinetic assay to show that the N terminus of BO is translocated cotranslationally, indicating that the first transmembrane segment is inserted cotranslationally (20). In this study, we examine the translocation order of other extracellular domains of the protein and the timing of translocation with respect to translation.…”

“…BR synthesis was induced and quantified as described (20). For derivatives of the ⌬ura3 strain MPK406, the growth medium was supplemented with 50 g of uracil per ml.…”

“…For derivatives of the ⌬ura3 strain MPK406, the growth medium was supplemented with 50 g of uracil per ml. Full-length His-tagged BO was Ni 2ϩ -affinity-purified as previously described (20).…”

“…BO translocation and elongation were measured as described (20) with slight modifications to improve the assay. Briefly, cells expressing BO cysteine mutants were incubated in AMS, then radiolabeled with a pulse of 35 S-Met, followed with a nonradioactive Met chase.…”

“…The megaprimers were combined with 5Ј-TACAAGAC-CGAGTGGGGGACT-3Ј in the second PCR step. PCR products were digested with restriction enzymes, combined with the appropriate fragment encoding a C-terminal His-tag (20), and cloned in pMPK85, an H. salinarum integrating vector (22). Cysteine was substituted at position 116 by mutagenesis with the primer 5Ј-GCGCGGATCCGACGTGAAGA-3Ј and the degenerate primer 5Ј-GGCGGTTCAGATCATGATVVNGTCG-GCACCGACGAGCGCAA-3Ј (V ϭ A, C, or G).…”