The evolutionary emergence of organelles was a defining process in diversifying biochemical reactions within the cell and enabling multi-cellularity. However, compartmentalization also posed a great challenge - the need to import proteins synthesized in the cytosol into their respective sites of function. For example, one third of all cellular proteins must be targeted and translocated into the endoplasmic reticulum (ER), which serves as the entry site into the majority of endomembrane compartments. Decades of research have set down the fundamental principles of how proteins get from the cytosol into the ER. I will discuss our recent studies which have brought forward new pathways and have enabled better definition of the rules governing substrate recognition in the multifaceted and regulated process of protein targeting and translocation to the ER.