The Golgi apparatus is an essential eukaryotic organelle that plays crucial roles in biosynthesis, secretion, and endolysosomal homeostasis. As the nexus of biosynthetic and endocytic membrane traffic, the Golgi orchestrates a wide range of protein transport pathways that underpin cellular physiology. Beyond its sorting and transport functions, the Golgi performs post-translational processing and lipid synthesis, and has recently emerged as a key regulator of intracellular signaling pathways. Over 120 years of active research have produced a vast body of knowledge spanning key aspects of Golgi structure, organization, and function. Nevertheless, fundamental questions about the Golgi remain unanswered and subject to extensive debate and controversy. In this thesis, I investigate two of the most debated aspects of mammalian Golgi biology: a) the specific mechanisms by which Golgi resident proteins maintain their steady-state localization, and b) the mechanistic basis of secretory cargo transport through the Golgi stack. Informed by findings from the model organism S. cerevisiae, I established mammalian tissue culture systems to test whether core intra-Golgi transport mechanisms are evolutionarily conserved. The second chapter of this thesis provides compelling evidence for the conservation of a crucial intra-Golgi recycling pathway that maintains the steady-state localization of trans Golgi resident proteins. The third chapter makes a strong case for cisternal maturation as a conserved, primary mechanism of vectorial cargo transit through the Golgi.
