Date

December 2007

Document Type

Dissertation

Degree Name

Ph.D.

Department

Dept. of Physiology and Pharmacology

Institution

Oregon Health & Science University

Abstract

A major challenge of modern biology is elucidating the mechanisms used by polytopic membrane proteins to achieve native tertiary structure in living cells. This challenge is made more acute because of the increasingly recognized number of diseases caused by the inability of membrane proteins to fold correctly. Early events of polytopic protein folding occur in the membrane of the endoplasmic reticulum and are orchestrated by the ribosome-translocon complex, a >3MD multi-component machine that is responsible for correctly orienting lumenal and cytosolic domains and inserting transmembrane segments into the lipid bilayer. The core component of the translocon is the sec61af3y heterotrimer, hypothesized to serve as a passive conduit for translocation and integration via an axial translocation pore and a lateral integration passage. Polytopic proteins have been further hypothesized to independently insert individual TM segments, via the translocon, into the lipid bilayer prior to folding. Here we use a photocrosslinking approach where a photoactive probe is coupled to a modified aminoacyl tRNA engineered to read through a UAG (amber) stop codon. Amber codons are engineered via PCR into eDNA templates and serially truncated to represent progressive points during synthesis. Because these templates contain no termination stop codon, they remain attached to the ribosome, thereby generating a uniform cohort of integration intermediates. We first examine the biogenesis of a complete native polytopic protein, aquaporin (AQP) 4. A series of truncations of AQP4 with probes engineered in one of three consecutive residues in the center of each TM permit us to examine the interaction of each TM with sec61 alpha as it enters and traverses sec61a. This study reveals that each TM initially enters sec61 in a preferred orientation and is sequentially replaced in this primary site by the downstream TM. Further, TMs can enter into different molecular environments proximal to sec61 as synthesis progresses. As many as 4 TMs can reside in proximity to sec61a simultaneously. We then use a similar approach to examine the early interactions of a TM derived from the second transmembrane domain of the cystic fibrosis transmembrane conductance regulator (CFTR). Here we find that, unlike AQP4 TMs, CFTR TM8 has a prolonged initial contact with sec61 and maintains this proximity even after peptidyltRNA cleavage with puromycin. Additionally, this persistent interaction is due to the presence of a charged aspartate residue, D924, in the center of TM8. When energy is depleted by addition of apyrase, release of TM8 from sec61 alpha is significantly delayed. When D924 is replaced with valine, TM8 no longer demonstrates a persistent interaction and leaves the translocon sooner. A third study of full length CFTR using velocity centrifugation gradients reveals that full length CFTR remains in association with the ribosome-translocon complex after completion of synthesis and is slowly released. In oocytes, replacement of fresh oocytosol facilitates release as does the addition of NTPs. These studies demonstrate that multiple TMs of polytopic proteins can remain in the vicinity of sec61 prior to integration and that certain substrates can remain associated with sec61 alpha after release from the ribosome. This suggests that sec61 plays an active role in the biogenesis of membrane proteins and that integration of multispanning proteins is more complex than previously thought.

Identifier

doi:10.6083/M4T43R37

School

School of Medicine

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