GPI-anchored proteins: now you see 'em, now you don't.

Many cell surface proteins are attached to membranes via covalent glycosylphosphatidylinositol (GPI) anchors that are posttranslationally linked to the carboxy-terminus of the protein. Removal of the GPI lipid moieties by enzymes such as GPI-specific phospholipases or by chemical treatments generates a soluble form of the protein that no longer associates with lipid bilayers. We have found that the removal of lipid moieties from the anchor can also have a second, unexpected effect on the antigenicity of a variety of GPI-anchored surface molecules, suggesting that they undergo major conformational changes. Several antibodies raised against GPI-anchored proteins from protozoa and mammalian cells were no longer capable of binding the corresponding antigens once the lipid moieties had been removed. Conversely, antibodies raised against soluble (delipidated) forms reacted poorly with intact GPI-anchored proteins, but showed enhanced binding after treatment with phospholipases. In the light of these findings, we have reevaluated a number of publications on GPI-anchored proteins. Many of the results are best explained by lipid-dependent changes in antigenicity, indicating this might be a widespread phenomenon. Since many pathogen surface proteins are GPI-anchored, researchers should be aware that the presence or absence of the GPI lipid moieties may have a major impact on the host immune response to infection or vaccination.     

Cytogenetic darkroom magic: now you see them, now you don''t.

Customary procedures used to determine chromosomal inheritance do not disclose many of the chromosomal polymorphisms known to be present, resulting in uninformative families. The sequential printing of individual chromosomes presented here is a technique that has increased the number of informative families in our studies. This technique has revealed previously unseen heritable chromosome differences and allowed the designation of parental origin.     

Salmonella: Now you see it,now you don't

Diseases caused by Salmonella species are characterized by bacterial invasion of host cells. Salmonella invasion requires a genetic locus (inv) with homology to bacterial systems involved in specific protein export and organelle assembly. Until recently, the actual Salmonella invasion factors exported or assembled by the inv system remained unidentified. It now appears that Salmonella produces novel appendages upon contact with host cells. These appendages are transient, appearing and disappearing rapidly from the bacterial surface. Appendages are altered in strains unable to invade due to mutations within the inv/spa locus. Therefore, a role for the invasion locus has been identified, providing another example of bacterial pathogens responding to signals provided by the host cell surface.     

Host‐associated genomic differentiation in congeneric butterflies: now you see it,now you do not

Ecotypic variation among populations may become associated with widespread genomic differentiation, but theory predicts that this should happen only under particular conditions of gene flow, selection and population size. In closely related species, we might expect the strength of host‐associated genomic differentiation (HAD) to be correlated with the degree of phenotypic differentiation in host‐adaptive traits. Using microsatellite and Amplified Fragment Length Polymorphism (AFLP) markers, and controlling for isolation by distance between populations, we sought HAD in two congeneric species of butterflies with different degrees of host plant specialization. Prior work on Euphydryas editha had shown strong interpopulation differentiation in host‐adapted traits, resulting in incipient reproductive isolation among host‐associated ecotypes. We show here that Euphydryas aurinia had much weaker host‐associated phenotypic differentiation. Contrary to our expectations, we detected HAD in Euphydryas aurinia, but not in E. editha. Even within an E. aurinia population that fed on both hosts, we found weak but significant sympatric HAD that persisted in samples taken 9 years apart. The finding of significantly stronger HAD in the system with less phenotypic differentiation may seem paradoxical. Our findings can be explained by multiple factors, ranging from differences in dispersal or effective population size, to spatial variation in genomic or phenotypic traits and to structure induced by past histories of host‐adapted populations. Other infrequently measured factors, such as differences in recombination rates, may also play a role. Our result adds to recent work as a further caution against assumptions of simple relationships between genomic and adaptive phenotypic differentiation.     

Now you see him, now you don't: experience, not age, is related to reproduction in kittiwakes

In long-lived species, individuals can skip reproduction. The proportion of breeders affects population growth rate and viability, there is a need to investigate the factors influencing intermittent breeding. The theory predicts that if lack of experience is an important constraint, breeding probabilities should increase with experience for individuals of the same age, whereas under the so-called restraint hypothesis, breeding probabilities should increase with age regardless of experience. However, because the probability of detecting individuals in the wild is generally less than 1, it is difficult to know exactly the number of previous breeding episodes (breeding experience). To cope with this issue, we developed a hidden process model to incorporate experience as a latent state possibly influencing the probability of breeding. Using a 22-year mark-recapture dataset involving 9970 individuals, we analysed simultaneously experience and age effects on breeding probabilities in the kittiwake (Rissa tridactyla). We did not detect an influence of age on adult breeding probabilities. We found that inexperienced birds breed less frequently than experienced birds. Our approach enables us to highlight the key role of experience on adults breeding probabilities and can be used for a wide range of organisms for which detection is less than 1.     

A systematic approach to pair secretory cargo receptors with their cargo suggests a mechanism for cargo selection by Erv14

The endoplasmic reticulum (ER) is the site of synthesis of secreted and membrane proteins. To exit the ER, proteins are packaged into COPII vesicles through direct interaction with the COPII coat or aided by specific cargo receptors. Despite the fundamental role of such cargo receptors in protein traffic, only a few have been identified; their cargo spectrum is unknown and the signals they recognize remain poorly understood. We present here an approach we term "PAIRS" (pairing analysis of cargo receptors), which combines systematic genetic manipulations of yeast with automated microscopy screening, to map the spectrum of cargo for a known receptor or to uncover a novel receptor for a particular cargo. Using PAIRS we followed the fate of ～150 cargos on the background of mutations in nine putative cargo receptors and identified novel cargo for most of these receptors. Deletion of the Erv14 cargo receptor affected the widest range of cargo. Erv14 substrates have a wide array of functions and structures; however, they are all membrane-spanning proteins of the late secretory pathway or plasma membrane. Proteins residing in these organelles have longer transmembrane domains (TMDs). Detailed examination of one cargo supported the hypothesis that Erv14 dependency reflects the length rather than the sequence of the TMD. The PAIRS approach allowed us to uncover new cargo for known cargo receptors and to obtain an unbiased look at specificity in cargo selection. Obtaining the spectrum of cargo for a cargo receptor allows a novel perspective on its mode of action. The rules that appear to guide Erv14 substrate recognition suggest that sorting of membrane proteins at multiple points in the secretory pathway could depend on the physical properties of TMDs. Such a mechanism would allow diverse proteins to utilize a few receptors without the constraints of evolving location-specific sorting motifs.     

Visualization of cargo concentration by COPII minimal machinery in a planar lipid membrane

Selective protein export from the endoplasmic reticulum is mediated by COPII vesicles. Here, we investigated the dynamics of fluorescently labelled cargo and non‐cargo proteins during COPII vesicle formation using single‐molecule microscopy combined with an artificial planar lipid bilayer. Single‐molecule analysis showed that the Sar1p–Sec23/24p‐cargo complex, but not the Sar1p–Sec23/24p complex, undergoes partial dimerization before Sec13/31p recruitment. On addition of a complete COPII mixture, cargo molecules start to assemble into fluorescent spots and clusters followed by vesicle release from the planar membrane. We show that continuous GTPase cycles of Sar1p facilitate cargo concentration into COPII vesicle buds, and at the same time, non‐cargo proteins are excluded from cargo clusters. We propose that the minimal set of COPII components is required not only to concentrate cargo molecules, but also to mediate exclusion of non‐cargo proteins from the COPII vesicles.     

Transport through the Golgi apparatus by rapid partitioning within a two-phase membrane system

The prevailing view of intra-Golgi transport is cisternal progression, which has a key prediction--that newly arrived cargo exhibits a lag or transit time before exiting the Golgi. Instead, we find that cargo molecules exit at an exponential rate proportional to their total Golgi abundance with no lag. Incoming cargo molecules rapidly mix with those already in the system and exit from partitioned domains with no cargo privileged for export based on its time of entry into the system. Given these results, we constructed a new model of intra-Golgi transport that involves rapid partitioning of enzymes and transmembrane cargo between two lipid phases combined with relatively rapid exchange among cisternae. Simulation and experimental testing of this rapid partitioning model reproduced all the key characteristics of the Golgi apparatus, including polarized lipid and protein gradients, exponential cargo export kinetics, and cargo waves.     

Beyond Atg8 binding: The role of AIM/LIR motifs in autophagy

Selective macroautophagy/autophagy mediates the selective delivery of cytoplasmic cargo material via autophagosomes into the lytic compartment for degradation. This selectivity is mediated by cargo receptor molecules that link the cargo to the phagophore (the precursor of the autophagosome) membrane via their simultaneous interaction with the cargo and Atg8 proteins on the membrane. Atg8 proteins are attached to membrane in a conjugation reaction and the cargo receptors bind them via short peptide motifs called Atg8-interacting motifs/LC3-interacting regions (AIMs/LIRs). We have recently shown for the yeast Atg19 cargo receptor that the AIM/LIR motifs also serve to recruit the Atg12–Atg5-Atg16 complex, which stimulates Atg8 conjugation, to the cargo. We could further show in a reconstituted system that the recruitment of the Atg12–Atg5-Atg16 complex is sufficient for cargo-directed Atg8 conjugation. Our results suggest that AIM/LIR motifs could have more general roles in autophagy.     

Regulated motion of glycoproteins revealed by direct visualization of a single cargo in the endoplasmic reticulum

The quality of cargo proteins in the endoplasmic reticulum (ER) is affected by their motion during folding. To understand how the diffusion of secretory cargo proteins is regulated in the ER, we directly analyze the motion of a single cargo molecule using fluorescence imaging/fluctuation analyses. We find that the addition of two N-glycans onto the cargo dramatically alters their diffusion by transient binding to membrane components that are confined by hyperosmolarity. Via simultaneous observation of a single cargo and ER exit sites (ERESs), we could exclude ERESs as the binding sites. Remarkably, actin cytoskeleton was required for the transient binding. These results provide a molecular basis for hypertonicity-induced immobilization of cargo, which is dependent on glycosylation at multiple sites but not the completion of proper folding. We propose that diffusion of secretory glycoproteins in the ER lumen is controlled from the cytoplasm to reduce the chances of aggregation.