Cellular membranes differ in protein and lipid composition as well as in the protein-lipid ratio. Thus, progression of membranous organelles along traffic routes requires mechanisms to control ...bilayer lipid chemistry and their abundance relative to proteins. The recent structural and functional characterization of VPS13-family proteins has suggested a mechanism through which lipids can be transferred in bulk from one membrane to another at membrane contact sites, and thus independently of vesicular traffic. Here, we show that SHIP164 (UHRF1BP1L) shares structural and lipid transfer properties with these proteins and is localized on a subpopulation of vesicle clusters in the early endocytic pathway whose membrane cargo includes the cation-independent mannose-6-phosphate receptor (MPR). Loss of SHIP164 disrupts retrograde traffic of these organelles to the Golgi complex. Our findings raise the possibility that bulk transfer of lipids to endocytic membranes may play a role in their traffic.
The discovery and optimization of biomolecules that reliably function in metazoan cells is imperative for both the study of basic biology and the treatment of disease. We describe the development, ...characterization, and proof-of-concept application of a platform for directed evolution of diverse biomolecules of interest (BOIs) directly in human cells. The platform relies on a custom-designed adenovirus variant lacking multiple genes, including the essential DNA polymerase and protease genes, features that allow us to evolve BOIs encoded by genes as large as 7 kb while attaining the mutation rates and enforcing the selection pressure required for successful directed evolution. High mutagenesis rates are continuously attained by trans-complementation of a newly engineered, highly error-prone form of the adenoviral polymerase. Selection pressure that couples desired BOI functions to adenoviral propagation is achieved by linking the functionality of the encoded BOI to the production of adenoviral protease activity by the human cell. The dynamic range for directed evolution can be enhanced to several orders of magnitude via application of a small-molecule adenoviral protease inhibitor to modulate selection pressure during directed evolution experiments. This platform makes it possible, in principle, to evolve any biomolecule activity that can be coupled to adenoviral protease expression or activation by simply serially passaging adenoviral populations carrying the BOI. As proof-of-concept, we use the platform to evolve, directly in the human cell environment, several transcription factor variants that maintain high levels of function while gaining resistance to a small-molecule inhibitor. We anticipate that this platform will substantially expand the repertoire of biomolecules that can be reliably and robustly engineered for both research and therapeutic applications in metazoan systems.
Fundamental questions regarding collagen biosynthesis, especially with respect to the molecular origins of homotrimeric versus heterotrimeric assembly, remain unanswered. Here, we demonstrate that ...the presence or absence of a single cysteine in type-I collagen's C-propeptide domain is a key factor governing the ability of a given collagen polypeptide to stably homotrimerize. We also identify a critical role for Ca
in non-covalent collagen C-propeptide trimerization, thereby priming the protein for disulfide-mediated covalent immortalization. The resulting cysteine-based code for stable assembly provides a molecular model that can be used to predict, a priori, the identity of not just collagen homotrimers, but also naturally occurring 2:1 and 1:1:1 heterotrimers. Moreover, the code applies across all of the sequence-diverse fibrillar collagens. These results provide new insight into how evolution leverages disulfide networks to fine-tune protein assembly, and will inform the ongoing development of designer proteins that assemble into specific oligomeric forms.
A defining feature of cells are membrane bilayers that separate them from their environment and, in eukaryotic cells, delineate intracellular organelles with specialized functions. In eukaryotes, the ...lipid composition of different organellar membranes varies both in terms of bulk and signaling lipids, lending these membranes different biochemical and biophysical characteristics. Yet most lipids are synthesized in the endoplasmic reticulum (ER), then redistributed asymmetrically from there via mechanisms that remain poorly understood. Lipid redistribution is mediated in part by lipid transport proteins that operate at membrane contact sites, where organelles are closely apposed but do not fuse, moving lipids between their membranes. Our lab and others are characterizing a new family of such proteins, called the VPS13-like proteins (or the repeating beta groove (RBG) domain family), that serve as channels for bulk lipid to flow between organelles, for example to facilitate membrane expansion. For my thesis project, my collaborators and I show that the protein SHIP164 shares structural and lipid transfer properties with this family of proteins and demonstrate that it localizes to early endocytic vesicles and contributes to their growth. Our findings raise the intriguing possibility that protein-mediated bulk lipid transfer could play a role in endosomal trafficking and maturation and support that non-vesicular protein-mediated lipid transfer is much more ubiquitous than previously appreciated.
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