Membrane proteins catalyse essential functions within the cell making them attractive drug target candidates. However, the general principles of membrane protein targeting, insertion and folding are less understood. Universally conserved membrane protein YidC is a key player in this pathway; it performs the dual function of assisting the clearance of Sec dependent proteins and inserting a subset of membrane proteins independently. In this study, we tested the involvement of the translocated substrate periplasmic N-tail in specifying its membrane protein translocase requirement using a bitopic model protein Pf3-Lep which does not require YidC or Sec for its translocation. On systematically increasing the length of its N-tail with neutral spacer peptides, we found that there is a certain N-tail length requirement (∼35 resides) beyond which YidC translocase is required. N-tails longer than 50 residues required both YidC and Sec for its translocation, however translocation was severely impaired beyond 60 residues N-tail length. N-tail translocation of larger periplasmic domains were studied using Maltose-Binding Protein and Alkaline Phosphatase fusions to the N-tail of Pf3-Lep. Surprisingly, these bulky N-terminal regions could be translocated by YidC-Sec even in the absence of their respective signal sequences in the N-terminal direction. These results hint at the involvement of chaperons and other membrane-associated components like SecA in keeping the periplasmic region in an unfolded state and facilitating the translocation of larger N-tails. Finally, we tested the role of charges on Pf3-Lep N-tail as translocase determinants and found that crowding of positive or negative charges led to YidC-Sec dependence. This study provides fundamental information regarding the translocation capacity of YidC and YidC-Sec mode of insertion of single-spanning membrane proteins in E.coli.