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Membrane protein insertion in both prokaryotic and eukaryotic organisms is a remarkably conserved process in all domains of life. SecYEG is the primary translocase in the cell as it is responsible for translocating the majority of the substrates through the membrane. A subset of proteins insert independent of the SecYEG translocase and insert by the YidC only pathway. YidC, which serves as the major focus of this dissertation, can play many different roles within the cell, as an independent insertase, or function in concert with the Sec translocase. The work presented in this dissertation is primarily focused on understanding the factors that determine the protein translocation pathway, as well as the mechanism by which YidC carries out its function.Initially we investigate the M13 procoat (PC) as a model substrate for membrane protein translocation. During membrane biogenesis, the procoat protein is inserted into the lipid bilayer in a strictly YidC-dependent manner. Here, we find that the translocase requirements can be altered for PClep in a predictable manner by changing the polarity and charge of the peptide region that is translocated across the membrane. This study provides a framework to understand why the YidC and Sec machineries exist in parallel and demonstrates that the YidC insertase has a limited capacity to translocate a peptide chain on its own.We further tested this polarity/charge hypothesis by showing that procoat-Lep insertion can become increasingly YidC/Sec dependent by making the periplasmic loop highly polar in the absence of any charged residues. Moreover, adding a number of hydrophobic amino acids to a highly polar loop can decrease the Sec-dependence of the otherwise strictly Sec-dependent membrane proteins. We also provide evidence that the increased Sec requirements for translocation of positively charged residues compared to negatively charged residues is not due to the pmf hindering translocation of positive charges. Taken together, the results support the polarity/charge hypothesis as a determinant for the translocase requirement for procoat insertion.Finally we examine the functional importance of various structural domains and residues based on the recent crystal structure of YidC2 from Bacillus halodurans. The structure revealed that the conserved positively charged residue within transmembrane (TM) segment one is located in a hydrophilic groove, embedded in the inner leaflet of the lipid bilayer. Here, we investigated the importance of the conserved positively charged residue for the function the E. coli YidC, S. mutans YidC2, and the chloroplast A. thaliana Alb3. The conserved arginine was required for the function of the Gram-positive S. mutans YidC2 and was necessary to complement the E. coli YidC depletion strain and to promote insertion of a YidC-dependent membrane protein. In contrast, the conserved positively charged residue was not required for the E. coli YidC or the A. thaliana Alb3. In addition, our results show that the C-terminal half of the helical hairpin is important for the activity of YidC since various deletions in the region either eliminate or impair YidC function. Interestingly, the precise sequence of amino acids of the hairpin was not required for function.