Eukaryotic cells are compartmentalised into membrane-enclosed organelles that perform specific functions. Proteins that are destined for the ER (endoplasmic reticulum) contain stretches of hydrophobic amino acids that act as signal sequences and may be located at either their N- or C-terminus, or within the polypeptide. The signal sequences of the majority of ERtargeted proteins are recognised by SRP (signal recognition particle), and are delivered to the ER where they translocate across its membrane co-translationally. Tail-anchored proteins, which possess a signal sequence at their C-terminus, must be delivered to the ER posttranslationally, and therefore use an alternative set of cytosolic components that collectively constitute the TRC40 pathway. A second group of precursor proteins that are delivered to the ER post-translationally are the short secretory proteins, which have also been proposed to use the TRC40 pathway. Alternative pathways that are also capable of delivering both tail-anchored proteins and short secretory proteins to the ER have been proposed, but a complete understanding of the biogenesis of these two groups is still lacking. Once at the ER, proteins must either fully translocate into the ER lumen (i.e. secretory proteins) or integrate into the ER membrane (i.e. transmembrane proteins). The Sec61 complex is primarily responsible for performing these actions, with the notable exception of tail-anchored proteins, which are instead proposed to integrate via the WRB/CAML complex. Through the use of mutagenesis, structural analysis, and newly discovered Sec61 inhibitors, these processes are beginning to be understood at a molecular level. In the following studies, I use an in vitro system to study the processes of both ER targeting and ER translocation. Firstly, by interfering with components of known ER delivery pathways, I highlight an underappreciated role for SRP in directing tail-anchored proteins to the ER posttranslationally, and demonstrate that, together, SRP and TRC40 account for almost the entirety of tail-anchored protein integration in vitro. Meanwhile, I show that short secretory proteins do not need TRC40 to be delivered to the ER, but are instead affected in a partial and substratespecific manner when the SRP pathway is disrupted. Secondly, I characterise the mycobacterial toxin mycolactone as a Sec61 inhibitor by showing a potent loss of ER translocation for cotranslationally translocated secretory proteins. Thirdly, I use mycolactone as a biochemical tool to analyse differences between Sec61-dependent substrates. Here, I find that short secretory proteins are inhibited to a lesser extent than co-translationally translocated secretory proteins, and observe differences between single-pass transmembrane proteins that correlate with how they are proposed to initially engage with the Sec61 translocon.