Protein targeting and translocation in Escherichia coli: LacZ fusions reveal a function for proto-oncogene homologues, a new effect of antibiotics and details of outer membrane biogenesis

Protein translocation is a fundamental process common to all living cells. It is essential for the delivery of extra-cytoplasmic proteins to their final destination. Facilitating this process is an evolutionarily-conserved protein complex called Sec complex in prokaryotes. The core Sec complex of Escherichia coli consists of the membrane proteins SecYEG, where SecY forms the main channel. Most secreted proteins are translocated post-translationally while some, primarily inner membrane proteins, are translocated while they are synthesized on the ribosome. In both cases, extra-cytoplasmic proteins are initially directed to the Sec machinery via their amino-terminal signal sequence. When the signal sequence of the outer membrane protein LamB is fused to the cytoplasmic reporter protein LacZ, the resulting hybrid is targeted for post-translational translocation. However, rapid folding of the LacZ segments in the cytoplasm prevents efficient translocation of the hybrid resulting in jamming of SecY.;The secretion stress exerted by the LamB-LacZ hybrid results in sensitivity to its inducer, maltose. Our lab previously isolated dominant mutations in cpxA that suppress this sensitivity. These mutations, called cpxA*, result in activation of an envelope stress response that results in translocation of LamB-LacZ to the periplasm. The next question was the identity of the Cpx-regulated factor(s) that enabled translocation of the hybrid. The first part of this thesis describes the identification and activity of YccA, a Cpx-induced protein capable of relieving LacZ hybrid jamming.;It has long been thought that the toxicity of the LamB-LacZ hybrid was due to jamming of the Sec machinery preventing secretion. We have shown that the toxicity is actually due to proteolytic destruction of SecY by the highly conserved protease, FtsH. We believe that folded LacZ sequences do not simply jam SecY but in fact slow translocation of LamB-LacZ greatly. Results presented in this work suggest that cells sense these struggling translocators and destroy them. Furthermore, our work shows that increased levels of YccA protect SecY from degradation by FtsH.;Interestingly, YccA is homologous to human Bax Inhibitor-1 (BI-1), an anti-apoptotic protein that restrains the activity of the tumor-suppressor, Bax. BI-1 is over-expressed in various types of cancer. Despite its obvious importance, the mechanism of BI-1 action remains unknown. We hope that further elucidating the YccA quality control mechanism will shed light on the function of this important cell death regulator in humans.;Agents that inhibit translation elongation result in translocators that are jammed by co-translationally translocated proteins fused to a ribosome by an unhydrolyzed tRNA. This report describes how antibiotics that block translation elongation cause proteolytic destruction of SecY while antibiotics that affect other stages of gene expression do not. Our results reveal a new activity for several old antibiotics and may help explain a decades-old puzzle of why antibiotics that inhibit translation elongation exhibit bactericidal effects much sooner in some bacteria than in others.;Lastly, this report describes biochemical evidence for coordination between the Sec complex and the beta-barrel assembly machinery (BAM) complex. The BAM complex of the outer membrane is responsible for assembling proteins into the outer membrane. Like the Sec complex, components of the BAM complex are highly conserved and found in mitochondria and chloroplasts. We show that, similar to protein targeting in mitochondria, the targeting of proteins to the outer membrane of E. coli via the Sec and BAM complexes occurs concurrently.;There are many powerful tools at our disposal for studying bacteria, especially E. coli, the most widely-used organism in molecular genetics. Given the remarkable conservation of many cellular processes, it seems only logical to make use of such a vast arsenal of tools, not to mention the encyclopedia of knowledge already available about model organisms, when attempting to decipher a disease process. This work provides an example of how bacteria can be used to investigate many problems, such as cancer, that might otherwise seem to have little connection to them.